Method for optical detection of an adjoining of a material component to a sensor material with the aid of biological, chemical or physical interaction and device for carrying out said method (variants)

ABSTRACT

Method for detecting biological or chemical components in liquid or gas is based on measuring changes of the sensor layer thickness due to binding reactions. A plate or a gap with two surfaces of a solid optical material is used as the sensor layer. The surfaces are located at a distance of more than 10 μm, which allows pumping liquids through the gap at moderate pressure drops and investigating large biological objects (e.g., cells), or employment of affordable plates that are rigid enough without any substrate. The indicated thickness of the plate or the gap permits using of ultra bright superluminescent diodes as light sources, because it allows recording within their narrow spectrum a sufficient number of interference maxima and minima for precise registration of molecular binding reactions, which lead to much higher sensitivity of the method as compared with thin-film methods.

TECHNICAL FIELD

The proposed invention refers to the field of development of methods andtools for biological and chemical analyses.

BACKGROUND ART

An analogue of the proposed method [U.S. Pat. No. 4,558,012, Int. Cl.G01N 33/54, U.S. Cl. 436/501, 1985] is known that is intended fordetection of chemical material components and measuring theirconcentration by detection of their binding to a sensor layer, whichcomprises:

-   -   irradiating of the sensor layer by light of various wavelengths,        for which the sensor layer is transparent, at least, partially;    -   registration in the reflected light of a signal, which depends        upon optical thickness of the said sensor layer and is due to        the fact that interference on the said sensor layer modulates        the reflection spectrum of the said sensor layer;    -   judging about the binding being detected from a change of the        said signal.

In this method, the sensor layer is formed on a non-metallic substratewith high optical absorption, made preferably of a semiconductor, darkglass or plastic. The sensor layer consists of a number of transparentdielectric layers, a material that binds the chemical substance beingdetected, and the said chemical substance itself that forms a thinnear-surface layer as a result of the said binding. The thickness of thesensor layer is chosen so that it acts as an antireflecting coating forpolychromatic light of wavelengths within the range of (525-600) nm,which is incident to the said sensor layer. Interference on the sensorlayer results in a reflection minimum in the said range. A change of thethickness of the sensor layer due to binding of the said chemicalsubstance results in a spectral shift of the said minimum and,consequently, a change of color of the reflected light. This colorchange is registered visually and used for judging about presence orconcentration of the chemical substance being detected.

Drawbacks of the analogue and the apparatus for its embodiment [U.S.Pat. No. 4,558,012, Int. Cl. G01N 33/54, U.S. Cl. 436/501, 1985] are itslow sensitivity, not sufficient reliability and low precision of theresults. This is due to qualitative, not quantitative, evaluation of theresult and subjective character of the visual evaluation of the colorchange. Besides, these method and apparatus do not permit real-timeregistration of binding of chemical substances and investigation ofkinetics of the process.

Another analogue [U.S. Pat. No. 4,820,649, Int. Cl. G01N 33/53, U.S. Cl.436/501, 1989] of the proposed method is known that is intended fordetecting components of biological systems, which comprises:

-   -   irradiating the sensor layer by light of various wavelengths,        for which the sensor layer is transparent, at least, partially;    -   registration in the reflected light of a signal, which depends        upon optical thickness of the said sensor layer and is due to        the fact that interference on the said sensor layer modulates        the reflection spectrum of the said sensor layer;    -   judging about the binding being detected from a change of the        said signal.

The method of the analogue [U.S. Pat. No. 4,820,649, Int. Cl. G01N33/53, U.S. Cl. 436/501, 1989] slightly differs from the method of theanalogue [U.S. Pat. No. 4,558,012, Int. Cl. G01N 33/54, U.S. Cl.436/501, 1985] in that it is applicable to substrates with highreflectivity, in particular, to metallic substrates. Matching theintensities of the light reflected from two boundary surfaces of thesensor layer, which is necessary for effective interference on thesensor layer and producing a clearly distinctive color, is achieved byemployment of a semitransparent reflective film of small metallicparticles. This metallic film is deposited onto the sensor layer afterbinding of the component being detected. This method and the apparatusfor its embodiment [U.S. Pat. No. 4,820,649, Int. Cl. G01N 33/53, U.S.Cl. 436/501, 1989] have the same drawbacks as the method and apparatus[U.S. Pat. No. 4,558,012, Int. Cl. G01N 33/54, U.S. Cl. 436/501, 1985].Moreover, they are also more complex and less reliable because of usingof the said metallic film.

The closest to the proposed method is a method of optical detection ofbinding of at least one material component to a substance located on asurface of or inside the sensor layer on the basis of a biological,chemical or physical interaction [DE 42 00 088 C2, Int. Cl. G01N 21/45,1997], which comprises:

-   -   irradiating the sensor layer by light of various wavelengths,        for which the sensor layer is transparent, at least, partially;    -   registration in the reflected or transmitted light of a signal,        which depends upon optical thickness of the said sensor layer        and is due to the fact that interference on the said sensor        layer modulates the reflection or transmission spectrum of the        said sensor layer, respectively;    -   recording the spectrum of the said reflected or transmitted        light as the said signal;    -   judging about the binding being detected from a change of the        said signal.

According to this method, the sensor layer is located on a sufficientlytransparent substrate and is irradiated by light of appropriatewavelengths from the side of the substrate. The sensor layer consistsof, at least, partially, a layer of transparent inorganic (e.g. oxides,nitrides) or organic polymer (e.g. polystyrene) and a substance thatimplements the binding to be detected. The said substance is located onthe surface of or inside the sensor layer and is capable to bind thesaid material component. Reaction of specific binding of an antibodywith an antigen can be mentioned as an example of such binding. Amaterial that enhances reflection is placed between the sensor layer andthe substrate. The material forms one boundary surface of the sensorlayer. The other surface is formed by an external medium. The externalmedium is commonly a biological solution under test, which contains orpresumably contains the said component, whose binding is the object ofdetection.

Interference on the sensor layer results from combining of two or moresecondary light waves produced as a result of partial reflection andpartial transmission on the boundary surfaces of the sensor layer and,probably, on the interface surfaces inside the sensor layer. Saidinterference modulates the reflection and transmission spectra of thesensor layer. The spectrum of the reflected or transmitted light isrecorded, and the absolute optical thickness of the sensor layer isdetermined from shape of the spectrum by analytical fitting. Informationabout a change of optical thickness of the sensor layer due to thebinding being detected and, consequently, about parameters of the saidbinding is obtained from a change of the recorded spectrum.

Unlike analogues [U.S. Pat. No. 4,558,012, Int. Cl. G01N 33/54, U.S. Cl.436/501, 1985] and [U.S. Pat. No. 4,820,649, Int. Cl. G01N 33/53, U.S.Cl. 436/501, 1989], the method-analogue [DE 42 00 088 C2, Int. Cl. G01N21/45, 1997] provides capability of real-time detection of binding ofmaterial components to a substance of the sensor layer and detachment ofthe said components from the substance of the sensor layer, which is animportant advantage of the method-analogue.

In the method-analogue, the sensor layer must be thin, i.e. there are anumber of limitations implied on its thickness:

-   -   the thickness is of the same order of magnitude as the        wavelength of the used light;    -   the double thickness is less than the coherence length of the        used light;    -   the thickness is within the range (0.3-10) μm, being not more        than 5 μm in important practical cases and 2 μm in preferable        variants.

The mentioned limitations are due to the principle of how the absolutethickness of the sensor layer is determined in the method-analogue.Employment of thicker sensor layers would result in interference patternwith many periods in the recorded spectrum. Unambiguous determination ofthe absolute thickness of the sensor layer from such interferencepattern would be difficult or impossible.

The mentioned principle and related limitations on thickness of thesensor layer give rise to a number of drawbacks of the method-analogue.The spectral dependence of intensity of the reflected or transmittedlight that serves for determination of the absolute optical thickness inthe method-analogue represents a smooth curve that slowly varies withinthe observed spectral range. Due to this fact, any intensity variationsin the recorded spectrum lead to significant errors in the measurementresults. This particularly refers to variations that are non-uniformover spectrum. They can arise from drifts of operating parameters of theradiation source, changes of its temperature, heating of opticalelements of the scheme, thermal and mechanical instabilities of theoptical scheme due to changes of ambient conditions, etc.

Application of the method-analogue to multi-channel registration ofstructural changes of substances of the sensor layer including bindingof material components to one or several substances of the sensor layeris known [U.S. Pat. No. 5,999,262, Int. Cl. G01B 9/02, U.S. Cl. 356/357,1999]. In this method, the said structural changes in several spatiallyseparated areas of the sensor layer are detected, and all the said areasare simultaneously irradiated by the light of the said wavelengths. Thespectrum of the said reflected or transmitted light is recorded for eachsaid area by using sequentially in time different wavelengths andimplementing the following operations for each of the said wavelengths:irradiating the said areas by monochromatic light of one wavelength andmeasuring intensity of the said reflected or transmitted light for eachsaid area.

As this takes place, the sensor layer is placed either on aplate-substrate, which is positioned on a base plate while measuring, ordirectly on the base plate. This is due to the fact that at themulti-channel registration [U.S. Pat. No. 5,999,262, Int. Cl. G01B 9/02,U.S. Cl. 356/357, 1999] the sensor layer is also thin, and the samelimitations as in the method-analogue [DE 42 00 088 C2, Int. Cl. G01N21/45, 1997] are implied on its thickness.

The method of multi-channel registration is based on analysis of form ofthe recorded spectrum in each channel and, accordingly, determining ofthe absolute optical thickness of the sensor layer in each area. Thismeans that the method-analogue [DE 42 00 088 C2, Int. Cl. G01N 21/45,1997] is used for every channel (each area of the sensor layer understudy). In this case, all the mentioned drawbacks of the method-analogueremain in its multi-channel variant. Moreover, they manifest themselveseven stronger. Since in the method of multi-channel registrationdifferent spectral regions are recorded sequentially in time, any driftsand instabilities in intensity of the analyzed light in whole or in anyregion of its spectrum result in lower accuracy of measurements.Besides, as the multi-channel registration requires a powerful lightsource, the negative role of thermal instabilities of all elements ofthe optical scheme, namely: the source, detector, dispersion elements orspectral filters, assembly elements, etc. sharply increases. Suchinstabilities cause not only drifts of spectral distribution of thelight intensity in each channel, but also drifts of intensitydistribution of the analyzed light over the channels. Amongcharacteristic examples, one can mention a change of color temperatureof the light source due to heating of a filament and a change ofintensity distribution of exposure along surface of the sensor layer andalong surface of the photodetector, where images of different areas ofthe sensor layer are transformed, due to the filament sag because ofheating. In these cases, uncontrollable drifts of both spectrum of theanalyzed light and intensity distribution over the registration channelstake place.

All this leads to low sensitivity, insufficient resolution, lowreliability and precision of results obtained by the method-analogue [DE42 00 088 C2, Int. Cl. G01N 21/45, 1997] and especially itsmulti-channel variant [U.S. Pat. No. 5,999,262, Int. Cl. G01B 9/02, U.S.Cl. 356/357, 1999]. Complexity, high labor input and cost can bementioned among drawbacks of the method and, even more, itsmulti-channel variant.

Variants of the apparatus that realizes the proposed variants of themethod are proposed.

The closest to the proposed apparatus is an apparatus-analogue intendedfor optical detection of binding of at least one material component to asubstance located on a surface of or inside the sensor layer on thebasis of a biological, chemical or physical interaction [DE 42 00 088C2, Int. Cl. G01N 21/45, 1997], which comprises:

-   -   a sensor layer;    -   a source of light, which irradiates the sensor layer, of        wavelengths that include at least operating wavelengths, for        which the sensor layer is transparent, at least, partially;    -   a detector, which is placed on the pathway of the light        reflected from the sensor layer or transmitted through the        sensor layer, for measuring the light intensity of operational        wavelengths in the spectrum of the received light;    -   a block of result generation, for example, a computer, to        generate information about the binding being detected on the        basis of changes of the said spectrum, whose input is connected        to the output of the detector.

In the apparatus-analogue, the sensor layer is located on a transparentsubstrate, which is made preferably of glass, and the light from thesource irradiates the sensor layer from the substrate's side. The sensorlayer comprises a transparent, preferably inorganic, optical substanceand a substance that binds a detected material component on the surfaceof or inside the sensor layer. A spectrometer preferably made on thebasis of a photodiode array is used as a detector to register reflectionor transmission spectrum of the sensor layer. The block of the resultgeneration in the apparatus-prototype is made capable to determine theabsolute thickness of the sensor layer from the recorded spectrum thatis modulated by interference on the sensor layer.

As was discussed above during consideration of the method-analogue [DE42 00 088 C2, Int. Cl. G01N 21/45, 1997], operation of theapparatus-analogue is based on the fact that binding being detected ofmaterial components on the surface of or inside the sensor layer changesoptical thickness of the sensor layer. The block of result generation inspecified moments of time determines the absolute optical thickness ofthe sensor layer from the spectrum recorded by a detector, and informsabout the binding being detected based on a change of this absolutethickness.

The used in the apparatus-analogue principle of determining of absolutethickness imposes a number of simultaneous restrictions on thickness ofthe sensor layer:

-   -   thickness is of order of magnitude of the wavelength of the used        light;    -   double thickness is less than the coherence length of the used        light;    -   thickness is in the range (0.3-10) μm, in practically important        cases and preferable variants not exceeding 5 μm and 2 μm,        respectively.

The mentioned peculiarities and restrictions of the apparatus-analoguegive rise to a number of drawbacks discussed earlier while analyzing thecorresponding method, namely, significant errors in measurement resultsdue to uncontrollable intensity variations over the recorded spectrum orits intervals, low sensitivity and resolution, low precision andinsufficient reliability of results of measurements.

A multi-channel variant of the apparatus-analogue is also known [U.S.Pat. No. 5,999,262, Int. Cl. G01B 9/02, U.S. Cl. 356/357, 1999], whichis intended for investigation of structural changes, in particular,binding of material components, in several areas of the sensor layer. Inthe said apparatus, a source is monochromatic and tunable; the sensorlayer is arranged either on a carrier plate or on a substrate placed onthe carrier plate for measurements; the light irradiates simultaneouslyall the areas under study; a detector represents a set of photoelectricdetectors; a control link is introduced between the block of resultgeneration and the source to switch the latter to another wavelength ofirradiated light after measurement by the detector of the intensity ofreceived light for each said area at one wavelength; the block of theresult generation is made capable to generate a spectral distribution ofintensity of the received light over wavelength for each said area;judgment about binding being detected is made based on a change of thesaid spectral distribution for each said area.

Principle of operation of this apparatus consists in point-by-pointdetermination of the spectrum for each area under study. At first,intensity in the spectrum is measured for all areas at one wavelength,then at another wavelength, etc. One determines absolute thickness ofthe sensor layer from the obtained spectra for each area and judgesabout binding being detected from a change of this thickness for eacharea.

A microtiter plate is also known [U.S. Pat. No. 6,018,388, Int. Cl. G01N21/03, U.S. Cl. 356/246, 2000] for multi-channel registration of bindingprocesses, which is a base component of the apparatus [U.S. Pat. No.5,999,262, Int. Cl. G01B 9/02, U.S. Cl. 356/357, 1999]. In this plate, asensor layer is up to 1 μm thick and formed on a bottom plate. There isat least one more layer between the sensor layer and the bottom plate.Back side of the bottom plate has an anti-reflecting coating. Areas ofthe sensor layer, for which the said binding is studied, are formed by anon-detachable joint of the bottom plate and a second plate that has anumber of holes. The sensor layer in each area forms the bottom of areaction cell while the holes of the second plate form side walls ofthese reaction cells.

Mentioned variants of the apparatus [U.S. Pat. No. 5,999,262, Int. Cl.G01B 9/02, U.S. Cl. 356/357, 1999 and U.S. Pat. No. 6,018,388, Int. Cl.G01N 21/03, U.S. Cl. 356/246, 2000] employ the same principle ofdetermination of thickness of the sensor layer as the apparatus-analogue[DE 42 00 088 C2, Int. Cl. G01N 21/45, 1997] and impose the samerestrictions on the thickness of the sensor layer. As it was discussedabove during analysis of the method [U.S. Pat. No. 5,999,262, Int. Cl.G01B 9/02, U.S. Cl. 356/357, 1999], all drawbacks of theapparatus-analogue [DE 42 00 088 C2, Int. Cl. G01N 21/45, 1997] areinherent to the apparatus [U.S. Pat. No. 5,999,262, Int. Cl. G01B 9/02,U.S. Cl. 356/357, 1999], including realization [U.S. Pat. No. 6,018,388,Int. Cl. G01N 21/03, U.S. Cl. 356/246, 2000], and they are even morepronounced.

Thus, the required technical result is to make measurement resultsindependent from uncontrollable variations of intensity of the analyzedlight in whole as well as in some parts of the spectrum and some regionsof the sensor layer surface, and, consequently, to increase accuracy andreliability of the measurements, sensitivity and resolution withsimultaneous reduction of a number of necessary operations, decreasingof labor-input and cost of the method and apparatus in both single- andmulti-channel variants including real-time operation.

DISCLOSURE OF INVENTION

To achieve the said technical result the first variant of a method ofoptical detection of binding of at least one material component to asubstance located on a surface of or inside a sensor layer due to abiological, chemical or physical interaction is proposed, whichcomprises:

-   -   irradiation of the sensor layer by light of various wavelengths,        for which the sensor layer is transparent, at least, partially;    -   registration in the reflected or transmitted light of a signal,        which depends upon optical thickness of the said sensor layer        and is due to the fact that interference on the said sensor        layer modulates the said reflection or transmission spectrum of        the said sensor layer, respectively;    -   recording the spectrum of the said reflected or transmitted        light as the said signal;    -   judging about the binding being detected from a change of the        said signal,        -   which is similar to the method-analogue.

The proposed method is characterized in that:

-   -   thickness of the sensor layer exceeds 10 μm and, at the same        time, exceeds the maximal wavelength of the said recorded        spectrum by at least one order of magnitude;    -   the light, which irradiates the sensor layer, is collimated.

Besides, in the recorded spectrum at least two maximums or minimums dueto the said interference are observed, and information about the bindingbeing detected is obtained from a spectral shift of the said maximums orminimums.

Besides, monochromatic light is used as the light that irradiates thesensor layer, its wavelength being tuned.

Besides, polychromatic light is used as the light that irradiates thesensor layer.

Besides, polychromatic light with continuous spectrum and coherencelength that is less than the double thickness of the sensor layer isused.

Besides, the sensor layer is placed on the substrate temporarily for thetime of the said signal registration or permanently.

Besides, the light irradiates the sensor layer from the substrate'sside, the substrate being transparent, at least, partially, for the saidlight.

Besides, a plate with surfaces not adjacent to any substrate is used asthe sensor layer.

Besides, liquid being tested that contains or presumably contains abiological or chemical component, whose binding is the object ofdetection, is placed on one of the irradiated boundary surfaces of thesensor layer, while the other boundary surface is formed with using asubstance that provides closeness of reflection coefficients of bothboundary surfaces.

Besides, liquid is placed on both irradiated boundary surfaces of thesensor layer; the liquid being tested that contains or presumablycontains a biological or chemical component, whose binding is the objectof detection, is placed on at least one of the said boundary surfaces;liquid with the refractive index close to the refractive index of theliquid under test is arranged on the other surface.

Besides, the liquid being tested is placed on both said boundarysurfaces and the said binding is detected from the side of both saidboundary surfaces.

Besides, a layer of the liquid under test that contains or presumablycontains a biological or chemical component, whose binding is the objectof detection, is used as the sensor layer; the irradiated boundarysurfaces of this layer are formed using hard optical materials; bindingof the said component to at least one of the said boundary surfaces isdetected.

Besides, binding of at least one material component in several spatiallyseparated areas of the irradiated region of the sensor layer isdetected; the spectrum of the said reflected or transmitted light isrecorded for each said area; the said spectrum is used as the saidsignal for each said area.

Besides, the proposed method is characterized in that:

-   -   in each said area the sensor layer is formed by a plate with        surfaces not adjacent to any substrate;    -   the spectrum of the said reflected or transmitted light for each        said area is recorded by using sequentially in time different        wavelengths of the light, which irradiate the sensor layer, and        measuring the intensity of the said reflected or transmitted        light for each said area at each wavelength.

Besides, the proposed method is characterized in that:

-   -   in each said area the sensor layer is formed by a layer of the        liquid under test, which contains or presumably contains a        biological or chemical component, whose binding is the object of        detection; the irradiated boundary surfaces of the said layer        are formed with using of hard optical materials; binding of the        said component to at least one of the said boundary surfaces is        detected;    -   the spectrum of the said reflected or transmitted light for each        said area is recorded by using sequentially in time different        wavelengths of light, which irradiate the sensor layer, and        measuring the intensity of the said reflected or transmitted        light for each said area at each wavelength.

Besides, polychromatic light is used as the light that irradiates thesensor layer; the spectrum of the said reflected or transmitted lightfor each said area is recorded by using sequentially in time light ofdifferent wavelengths; the intensity of the said reflected ortransmitted light for each said area is measured for each saidwavelength.

Besides, all the said areas are irradiated simultaneously.

Besides, several different substances capable to selectively bindvarious material components are placed in the said areas.

Besides, bindings of the various material components to the saiddifferent materials are detected.

Let us explain the proposed variant of the method and show that it isits distinctive features that ensure the required technical result.

In this variant of the method, obtained results are independent ofuncontrollable variations of intensity of the analyzed light because ofusing the sensor layer with thickness that exceeds 10 μm and, at thesame time, exceeds the maximal wavelength of the said recorded spectrumby at least one order of magnitude. Besides, the wavelength range of thelight that irradiates the sensor layer is chosen rather wide; severalmaximums and minimums due to the interference on the sensor layer areobserved in the recorded spectrum; judgment about the binding under testis made from a spectral shift of the said maximums and minimums. Atleast two said maximums and minimums (claim 2) are observed in therecorded spectrum while in preferred realizations of the method multiple(5-10 and more) intensity maximums and minimums are observed. Thespectral position of the said maximums and minimums serve as the sourceof information about the binding being detected. Binding of materialcomponents to substances located on a surface of or inside the sensorlayer leads to an increase of optical thickness of the sensor layer d*,and, consequently, to a decrease of the period Δν of the periodicinterference pattern recorded in the frequency spectrum of the reflectedor transmitted light, according to the relationship:Δν=c/2d*  (1).

Here c is the speed of light in vacuum andd*=∫ ₀ ^(d) n(z)dz  (2),

where d is geometric thickness of the sensor layer, n(z) is thedistribution of the refraction index of the sensor layer over itsthickness.

It is easy to mention that the increase of optical thickness of thesensor layer results in a shift of the interference maximums andminimums in the spectrum of the reflected or transmitted light towardslonger wavelengths. Accordingly, the opposite reaction of detachment ofmaterial components from substances of the sensor layer is accompaniedby a decrease of optical thickness and to a shift of the interferencemaximums and minimums towards shorter wavelengths.

It is important to note that the light beam, which irradiates the sensorlayer and whose spectrum is recorded, should be sufficiently collimatedso that the difference of optical paths inside the sensor layer fordifferent rays of the beam and all wavelengths of the recorded spectrumdoes not exceed approximately a fourth of light wavelength. Otherwise,at this wavelength an interference maximum for one ray overlaps aninterference minimum of another ray, which causes diffusion of theinterference pattern in the spectrum of the reflected or transmittedlight. In multi-channel modifications of the method (see below), thesaid collimation level should be provided individually in each channelor in each area of the sensor layer, which is analyzed independentlyfrom other areas.

As the thickness of the sensor layer exceeds wavelengths of the recordedspectrum by one or more orders of magnitude, frequencies of the recordedspectrum significantly exceed the frequency period of the interferencepattern Δν, and maximums or minimums corresponding to larger values ofΔν are observed in the recorded spectrum. Hence, it is spectral shift ofthe interference maximums and minimums that is first of all observed ata change of thickness of the sensor layer, and this shift is a much morepronounced effect that a change of distance between maximums or minimumsin the recorded spectrum. When thickness of the sensor layer issufficiently large as compared with operating wavelengths, the spectrumof operating wavelengths is wide enough, and at most common in practicelow reflection coefficients of the boundary surfaces of the sensor layerthe interference pattern in the recorded spectrum has many periods andcan be considered with good accuracy as a sinusoid or, more precisely, asinusoid modulated by spectrum of the affecting light. In this case,tracking of phase of the said sinusoid can be a convenient method toobtain an information signal. To take into account the modulatedcharacter of the sinusoid, it is convenient to apply the fast Fouriertransformation to the recorded spectrum containing the interferencepattern and to track the phase of the first harmonic.

Thus, in the proposed method the information signal is formed on thebasis of registration of spectral position of individual interferencemaximums or minimums, or the whole interference pattern (a “comb” ofinterference maximums and minimums). Hence, contrasting to the knownmethod [DE 42 00 088 C2, Int. Cl. G01N 21/45, 1997 and U.S. Pat. No.5,999,262, Int. Cl. G01B 9/02, U.S. Cl. 356/357, 1999], variations ofintensity of the analyzed light in whole or of different parts of therecoded spectrum do not affect the information signal, which ensures therequired technical result.

The required technical result is also achieved in respect ofsimplification of the method, reduction of the number of necessaryoperations, decreasing of labor input and cost. Indeed, unlike the knownmethod [DE 42 00 088 C2, Int. Cl. G01N 21/45, 1997 and U.S. Pat. No.5,999,262, Int. Cl. G01B 9/02, U.S. Cl. 356/357, 1999], the proposedmethod does not require analytical fitting of regions of spectraldependences or getting information about absolute thickness of thesensor layer, although it is capable to do this. Generally speaking, theinformation about absolute thickness is excessive for registration ofreactions of binding of biological and chemical components to substancesof the sensor layer. For registration of reactions of binding anddetachment it is sufficient to register changes of thickness, i.e.measure relative, not absolute, quantities. On one hand, this is mucheasier, on the other hand, provides much higher precision. It is theprinciple that is used in the proposed method.

One of the options to register the spectrum modulated by interference onthe sensor layer is employment of monochromatic light as the light thatirradiates the sensor layer; its wavelength is tuned (claim 3) onwavelengths, for which the sensor layer is transparent, at least,partially. In this case, registration of the spectrum is implemented inthe simplest way, i.e. by measuring the intensity by, for example, aphotodetector, and spectral distribution of the light intensity can bemeasured at a highest resolution on both intensity and wavelength. Thus,the required technical result is ensured. Besides, this realization ofthe method is good for registration of binding in a large number ofdifferent areas of the sensor layer simultaneously, i.e. for obtainingthe required information in a large number of channels concurrently andindependently from each other.

Other options of spectrum registration are in using of polychromaticlight (claim 4) as the light that irradiates the sensor layer. Thesetechniques are most appropriate for single-channel schemes or schemeswith a small number of registration channels because of lowerrequirements to the radiation source, diminished number of operations,simplicity and lower cost. In these techniques, the spectrum of thereflected or transmitted light is recorded either entirely at a time,e.g. by an array spectrometer, or by measuring sequentially in timeintensity of the light at one wavelength for a number of operatingwavelengths. While affecting the sensor layer by polychromatic light itis reasonable to use the light with continuous spectrum and coherencelength that is less than the double thickness of the sensor layer (claim5). The continuity of the spectrum is important for finding the spectralposition of interference maximums or minimums with maximal accuracy, andthe mentioned condition for the coherence length means that there arenot less than two interference maximums or minimums of intensity of thetransmitted or reflected light within the spectrum width of usedpolychromatic light. Importance of the latter condition was alreadymentioned above. Strictly speaking, a less strict limitation could beimplied to obtain not less than two interference periods in the recordedspectrum. The coherence length that is less than the double optical, notgeometric, thickness of the sensor layer would be a sufficientcondition. It is known that the optical thickness always exceeds thegeometric one. The limitation mentioned in claim 5 means that there arealways more than two interference periods in the recorded spectrum.

It should be noted that in the proposed method the condition “thecoherence length of the light that irradiates the sensor layer is lessthan the double thickness of the sensor layer” does not excludeinterference or observation of the interference pattern in the recordedspectrum. This is easy to understand taking into account that recordingof the spectrum of the reflected or transmitted light while affectingthe sensor layer by polychromatic light is always done by extracting ofnarrow spectral intervals from the reflected or transmitted light neardifferent wavelengths and registration of the light intensity withinthese intervals. The light extracted in each said spectral interval hasthe coherence length, which is much longer than that one of the originalpolychromatic light and, at the same time, larger than the doublethickness of the sensor layer. The latter condition means that for eachsaid extracted narrow spectral interval the result of interference onthe sensor layer is observed in the recorded spectrum.

The proposed method allows for different options of formation of thesensor layer. At rather small thickness (tens of microns) the sensorlayer is formed on a substrate to provide the necessary mechanicalfirmness and stability of the measuring scheme. It is rational also touse the substrate while working with replaceable samples of the sensorlayer for reliable fixation of the said replaceable sample on the solidand firmly fixed substrate during a study of binding reactions in thesample. When the sensor layer is placed on the substrate temporarily forthe time of registration of the said signal or permanently (claim 6), itis preferable to irradiate the sensor layer by light from thesubstrate's side, the substrate being transparent, at least, partially,for the light (claim 7). This increases accuracy of measurements andenlarges field of application of the method, providing a possibility ofoperation with absorbing or diffusing media being tested, which, in thiscase, are placed with respect to the sensor layer from the side that isopposite to the substrate.

Since in the proposed method upper limits for thickness of the sensorlayer, which are inherent to the method-analogue and other analogues,are eliminated, there is an opportunity to employ a plate not adjacentto any substrate as the sensor layer (claim 8). Fabrication of thesensor layer on the basis of a relatively thick plate (e.g. hundreds ofmicrons) provides the necessary hardness of the sensor layer andstability of its characteristics without using of carrier or guideplates (substrates) and, hence, permits one to simplify the method andto reduce its cost. The plate that forms the sensor layer can beimmersed in the medium under test (liquid or gas), from whose side thebinding under investigation takes place. Another preferable variant isthat the plate that forms the sensor layer serves as the bottom or awall of a reaction cell, in which the medium under test (e.g. abiologically active solution) is placed.

In both cases, i.e. for the sensor layer located on a substrate orfabricated on the basis of a separate plate, the preferable realizationof the method is that one, in which the liquid under test containing orpresumably containing a biological or chemical component, whose bindingis the object of detection (the subject of investigation), is placed onone of the irradiated boundary surfaces of the sensor layer while theother boundary surface is formed with using of a substance that providescloseness of reflection coefficients of both boundary surfaces (claim9). For example, the sensor layer forms the bottom or a wall of areaction cell. Then when using a substrate, it is reasonable to form theother boundary surface of the sensor layer using an optically densersubstance so that the reflectivity of the interface between thissubstance and the sensor layer is almost equal to the reflectivity ofthe interface between the sensor layer and the liquid under test. Thesaid optically denser substance can be either the substance of thesubstrate or the substance of the layer specially introduced between thesensor layer and the substrate. In both cases, it is reasonable to usean antireflecting coating on the other, rear, surface of the substrateto suppress parasitic reflection. If the sensor layer is formed by aseparate plate, whose one surface contacts the liquid under test and theother surface contacts the air, then it is reasonable to form anantireflecting coating on the other surface of the plate. While thistakes place, in all described variants according to claim 9, closenessof reflection coefficients of both boundary surfaces of the sensor layeris ensured. This allows one to reach the maximal contrast of theinterference pattern in the spectrum of the reflected or transmittedlight and, hence, the maximal ratio of useful signal to backgroundsignal. Thus, sensitivity of the method and precision of measurementsincrease.

If the sensor layer is formed by a separate plate not adjacent to anysubstrate, it is possible to use liquid on the second boundary surfaceto equalize reflection coefficients of both boundary surfaces. For this,liquids are placed on both irradiated boundary surfaces of the sensorlayer; the liquid under test that contains or presumably contains abiological or chemical component, whose binding is the object ofdetection (claim 10), being placed on at least one of these boundarysurfaces. In the simplest and most preferable realizations, whenrefractive indices of materials near both boundary surfaces of thesensor layer are equal, the liquids on both boundary surfaces shouldhave the same refractive index. For example, it may be the same liquidunder test. This opportunity can be realized by a special design of aflow cell, in which the liquid under test flows on both sides of theplate—sensor layer, or by simple immersion of the plate—sensor layerinto a reservoir with the liquid under test. The most preferablerealization is that, in which the liquid under test is placed on bothboundary surfaces and the said binding is detected from both saidboundary surfaces (claim 11). Such realization of the method doubles achange of the optical thickness of the sensor layer and, hence, doublessensitivity and resolution of the method while analyzing the liquidunder test for presence of any biological or chemical components.

The proposed method opens a number of new possibilities, when a layer ofthe liquid under test, which contains or presumably contains abiological or chemical component, whose binding is the object ofdetection, is used as the sensor layer; the irradiated boundary surfacesof the said layer are formed with using of hard optical materials; andbinding of the said component to at least one of the said boundarysurfaces is detected (claim 12). In particular, fabrication of thesensor layer is significantly simplified and, respectively, the wholemethod becomes much simpler and more cost-effective. Instead ofdeposition of the sensor layer onto a substrate or its fabrication as aplate, in this case it is sufficient to position two prepared flatsurfaces of optical blocks so that they face each other, fix thedistance between them by an insertion of appropriate thickness, andintroduce the liquid under test into the gap. Obviously, at registrationof the information signal in the transmitted light the optical materialson both sides of the layer of the liquid under test should besufficiently transparent, while at registration of the informationsignal in the reflected light transparency of only that optical block,from which the light is incident to the layer, is sufficient. Animportant circumstance is that the insertion material can be chosen sothat its thermal extension at temperature drifts corresponds to adecrease of the refractive index of the liquid under test. In such away, uncontrollable changes of optical thickness of the sensor layer dueto temperature drifts are compensated. Only the changes of opticalthickness are registered, which are due to binding components from theliquid under test to the substances (e.g. specifically bindingsubstances, bio-receptors, etc.) located on the said prepared surfacesof the optical blocks. As a result, measurement errors significantlydecrease, the accuracy and reliability of the results increase.

It should be noted that both variants of the proposed method accordingto claim 1 and claim 20, and claim 97, discussed below, apply also todetection of binding components of not only liquid components asdiscussed above, but also to gases. Liquids under test in the proposedmethod are solutions (including colloid mixtures, suspensions, etc.)that contain biological or chemical components, which, as a rule, arebiologically active. Among such biological components different antigensand antibodies, proteins, viruses, their fragments and antigendeterminants, bacteria, nucleic acids, their fragments and nucleotidesequences, lipids, polysaccharides, carbohydrates, enzymes, hormones,etc., and also receptors specific to these components. In the proposedmethod, binding of such components to substances located on a surface ofor inside an optically transparent sensor layer is registered. Thesesubstances can be optical substances that form boundary surfaces of thesensor layer or receptors for binding components being detected, whichare immobilized on these surfaces. Besides, the binding can take placeinside the sensor layer, for example, inside a three-dimensionalbiomolecular array (polymer long-chain molecules, dextrain,polypeptides, etc.) or in pores inside the sensor layer, wherecorrespondent receptors can be also immobilized. Similarly, the sensorlayer can adsorb or absorb detected components of gaseous mixtures onits surface or inside, for example, in its pores, including using ofspecifically sensitive substances (e.g. phthalocyanines for nitricoxide, etc.). As a result of binding of gaseous components and vapors,the sensor layer can also change the thickness (in particular, swell) orthe refractive index, or spectral characteristics oftransmission/reflection (color), etc. Appropriate substances for thesensor layers capable to bind biological and chemical components areknown from state of the art of biochemical and gas analyses.

The proposed method applies also to registration of binding ofcomponents on several or multiple channels, data for each registrationchannel being received independently of data from the other channels.The data are received for all the channels either simultaneously(parallel mode) or the channels are interrogated in sequence (serialmode). In some cases, groups of channels are interrogated in sequence(line-by-line mode). Combinations of the mentioned modes are alsopossible. In multi-channel modifications of the first variant of theproposed method binding of at least one component in several spatiallyseparated areas of the irradiated region of the sensor layer isdetected; the spectrum of the said reflected or transmitted light foreach said area is recorded; this spectrum is used as the said signal foreach said area (claim 13). In one registration channel either a singlearea is used, for which one receives a separate information signal (inthis case, the spectrum), or a group of areas, in which equal conditionsare created for binding components being detected. For example, oneregistration channel can be realized on the basis of one reaction cell,in which a receptor substance for binding a definite component isimmobilized. This channel corresponds to one said area when aninformation signal is registered from this cell as a whole, or toseveral areas, if independent information signals are obtained fromdifferent areas of the cell (e.g. if the light transmitted or reflectedfrom the cell is incident simultaneously on several photodetecting zoneswith independent outputs). To increase accuracy of measurements andreliability of results in the latter case it is reasonable to averagethe information signals over several areas within one reaction cell anduse the obtained result as an information output of this registrationchannel. However, one can also consider each area that corresponds to aseparate photodetecting zone and separate information output as forminga separate registration channel.

The said areas can be regions of surface of either a one-piece sensorlayer or a sensor layer that consists of separate pieces. In the lattercase, these pieces can be significantly spatially separated and evenhave different orientation of surface, e.g. if the sensor layer consistsof a number of regions with fiber-optical inputs and outputs. It is onlynecessary that each said area is irradiated by light and detection of aseparate independent information signal is provided for each area. Forthis, it is sufficient that no said area overlaps the light incident toanother area. Each said area comprises parts of both boundary surfacesand of volume of the sensor layer, which are irradiated by the samelight beams, including cases, when binding of components from bothboundary surfaces' side or inside the sensor layer is studied.

Multi-channel modifications of the proposed method are extremelyimportant and provide the said technical result for a number ofpractically important purposes. First of all, independent registrationof the information signal from several channels permits using channelsto form a reference signal (reference channels) for different purposes.For example, it is reasonable to use the reference channels tocompensate possible accidental errors, parameters' spread andnon-uniformity of samples containing or presumably containing thecomponents being bound.

In addition, the reference channels are applied to investigatespecificity of binding of biological or chemical components to thereceptors located on the surface of or inside the sensor layer. Forexample, for quantitative analysis of a solution for presence of abiologically active component (infectious or toxic agent, antigen orantibody, etc.), a reagent capable to selectively bind (recognize) thecomponent under study is immobilized on the surface of the sensor layeror inside it (e.g. in a three-dimensional biomolecular array). Presenceof this component in the solution is determined on the basis of theobtained data about its specific binding with the recognition reagent.To allow for non-specific (non-selective) binding of this or othercomponents with the surface or volume of the sensor layer the referencechannels are used.

Besides, during analyses that repeat in time or continuous monitoringthe reference channels can serve to take account of temperature driftsas well as other physical or chemical instabilities (e.g. pressure,density, pH of the solution or concentration of parasitic additions,etc.).

In all these tasks, as a rule, the reference channel is in the sameconditions as the information channel except conditions for selectivebinding of the component under study to the recognition reagent.

Employment of the reference channels lowers the registration threshold,increases reliability and accuracy of measurements and, thus, providesthe required technical result.

Another group of tasks, where multi-channel registration is required, isprovision of high throughput screening of the analyses. This isparticularly important, for example, for testing new pharmaceuticalproducts, when a huge number of tests are carried out to checkinteraction of the product with different reagents. For such tasks, inparticular, a realization of the proposed method with the number ofchannels in accordance with modem standards of immunoassays (e.g.immuno-ferment assay “ELISA”—Enzyme-Linked Immuno-Sorbent Assay) isreasonable. In this case, the channels correspond to reaction cells ofstandard microtiter plates that contain, for example, 96 (12×8 array),384 (24×16 array), 1536 (48×32 array) cells. The proposed method permitsinvestigation of binding of biological and chemical components (e.g.with different components being bound and/or different binding reagents)simultaneously (in parallel) in a large number of channels correspondingto the said spatially separated areas of the sensor layer. This stronglyincreases the productivity of the method, decreases its cost, providingthe said technical result.

The third group of tasks resolved with the multi-channel registration isrecognition of complex multi-component mixtures and analysis of presencein them of simultaneously several components. It is discussed below inconnection with claims 18 and 19.

One of the most preferable realizations of the multi-channelmodification of the first variant of the proposed method is that in eachsaid area the sensor layer is formed by a plate with surfaces notadjacent to any substrate; the spectrum of the said reflected ortransmitted light for each said area is recorded by using sequentiallyin time different wavelengths, which irradiate the sensor layer;intensity of the said reflected or transmitted light for each said areais measured at each of these wavelengths (claim 14). Scanning thewavelength of the light that affects the areas of the sensor layerprovides the simplest way to register the said spectrum for each saidarea. This is because in this case analysis of the reflected ortransmitted light represents measuring intensity of the light in eachmoment of time only at one wavelength, the spectrum wavelengths beingsequentially changed. Independence of the measurements of intensity foreach area is realized by projection of light from different areas ontodifferent photodetector zones with independent outputs, which form aphotodetector array, e.g. a CCD-array or a camcorder. To do this, animage of areas of the sensor layer is formed on the photodetector arrayor these areas are simply projected onto the said array by usedcollimated light. As a rule, the photodetector array is smaller than theset of areas under study, so optical systems are applied to form animage of smaller size or to project a collimated light beam of a reduceddiameter, which is reflected from the said areas of the sensor layer ortransmitted through them.

In this case, fabrication of the sensor layer as a plate not adjacent toany substrate realizes the method in the simplest way. It is convenientto form on this plate reaction cells correspondent to differentregistration channels by, for example, usual deposition (from solutionsor by sputtering) of recognition reagents on different regions of theplate, by etching wells, by gluing masks with holes, gluing or weldingthis plate with another plate, in which holes form the reaction cells,or by other methods.

The multi-channel method is also realized in a very simple way, when ineach said area the sensor layer is formed by a layer of the liquid undertest containing or presumably containing a biological or chemicalcomponent, whose binding is the object of detection; the boundarysurfaces of this layer exposed to the light are formed with hard opticalmaterials; binding of the said component to at least one of the saidboundary surfaces is detected; the spectrum of the said reflected ortransmitted light for each said area is recorded by using sequentiallyin time different wavelengths, which irradiate the sensor layer;intensity of the said reflected or transmitted light for each said areais measured at each of these wavelengths (claim 15). This case issimilar to the previous one with the only difference that reaction cellsare formed by holes in an insertion between two optical blocksrestraining the layer of the liquid under test (see comments to claim 12above).

Another realization of the method consists in that polychromatic lightof wavelengths, for which the sensor layer is sufficiently transparent,irradiates separated areas of the sensor layer. That is, the said areasare exposed to the polychromatic light; the spectrum of the saidreflected or transmitted light is recorded for each said area; therecorded spectrum is used as the said signal for each said area. Inparticular, at a small number of channels, each channel can be equippedby an array spectrometer. This significantly decreases the number ofnecessary operations and time consumption, makes the method simpler,lowers its cost and, in the same time, increases reliability of obtaineddata, which provides the said technical result. Another possibility thatis preferable for a large number of channels consists in thatpolychromatic light is used as the light that irradiates the sensorlayer; for each said area the spectrum of the said reflected ortransmitted light is recorded by using sequentially in time differentwavelengths; the intensity of the said reflected or transmitted lightfor each said area is measured at each of these wavelengths (claim 16).For example, the light near one wavelength is extracted from thetransmitted or reflected light by a dispersion element (a monochromator)or a spectral filter; at the said wavelength one registers an image ofthe said areas of the sensor layer on an array photodetector orrepresentation of the said areas produced on the array photodetector bycollimated light. By scanning the wavelength, the intensity distributionover wavelengths, i.e. the spectrum, is obtained for each area.

In realization of multi-channel modifications of the proposed method, itis preferable to affect by light simultaneously all areas of the sensorlayer, in which binding of the components is investigated (claim 17).This permits one to avoid scanning by light over the sensor layersurface, diminish the number of operations and time consumption,simplify the method and decrease its cost, increase reliability ofobtained data and accuracy of measurements, providing the said technicalresult. In addition, this makes possible simultaneous (parallel)registration in many channels and, thus, increases temporal resolutionof the method. This is important for observation of binding componentsin real time and investigation kinetics of this process.

All discussed multi-channel modifications of the proposed method allowone to effectively recognize complex multi-component mixtures anddetermine presence in them of simultaneously several components.

For this, several different substances capable to selectively bind (i.e.recognize) diverse material components are arranged on the saidspatially separated areas of the irradiated region of the sensor layer(claim 18). This engineering solution is rational also for investigationof interaction of one given component (e.g. new medicament) with a largenumber of different recognition substances simultaneously. Besides,binding of diverse material components to the said different substancesis detected (claim 19). In the simplest case, when selectivity ofbinding of each component with correspondent recognition reagent issufficiently high, not more than one reagent and not more than onecomponent under study correspond to each said area, i.e. one area “isresponsible” for recognition of one component, some areas being used asreference channels. In other cases, a complex pattern of signals isobtained from the said areas, each signal possessing low specificity toan individual component, but the whole pattern proves to be selective(similarly to a fingerprint) with respect to the whole mixture undertest. A mixture can be recognized, for example, by using computermethods of pattern recognition. Such modifications of the proposedmethod can be used for design of “biochips”, “gene chips”, electronic“nose” and “tongue”, etc.

Capability to recognize simultaneously several components in biologicalor chemical media and complex mixtures under test extends the field ofapplication of the method. Besides, this diminishes consumption of timeand resources to carry out analyses, providing the said technicalresult.

It should be noted that the proposed method provides capability ofreal-time registration and investigation of processes of binding(detaching) of components. This concerns also modifications of themethod, which include scanning over spectrum (sequential using ofdifferent wavelengths) of the affecting and either the reflected ortransmitted light, because the characteristic time of binding reactionsis usually sufficiently long (minutes and tens of minutes) and exceedsthe scanning time. Certainly, the proposed method permits detection ofresults of already completed reactions of binding (detaching) ofcomponents regardless the characteristic time of such reactions.

The second variant of the method of optical detection of binding of atleast one material component to a substance located on a surface of orinside a sensor layer due to a biological, chemical or physicalinteraction is also proposed, which comprises:

-   -   irradiation of the sensor layer by light of various wavelengths,        for which the sensor layer is transparent, at least, partially;    -   registration in the reflected or transmitted light of a signal,        which depends upon optical thickness of the said sensor layer        and is due to the fact that interference on the said sensor        layer modulates the said reflection or transmission spectrum of        the said sensor layer, respectively;    -   judging about the binding being detected from a change of the        said signal,        -   which is similar to the method-analogue.

The proposed method is characterized in that:

-   -   on the pathway of the light from the source to the detector a        scanned Fabry-Perot interferometer is placed along with the        sensor layer;    -   the base (i.e. optical path between the mirrors) of the scanned        Fabry-Perot interferometer is chosen sufficiently large so that        the period of the transmission spectrum of the said scanned        Fabry-Perot interferometer is at least twice smaller than the        width of spectral range of wavelengths participating in        formation of the recorded signal;    -   the base of the said scanned Fabry-Perot interferometer is        modulated;    -   distribution of intensity of the said reflected or transmitted        light, summed up over the said spectral interval, as a function        of base of the scanned Fabry-Perot interferometer is registered        as the said signal.

Besides, collimated light is used as the light that irradiates thesensor layer and the scanned Fabry-Perot interferometer.

Besides, polychromatic light with continuous spectrum is used as thelight that irradiates the sensor layer, the coherence length of the saidpolychromatic light and of the said reflected or transmitted light beingless than the double base of the scanned Fabry-Perot interferometer.

Besides, polychromatic light of the coherence length that is less thanthe double thickness of the sensor layer is used.

Besides, at least one intensity maximum due to correlation betweenspectral characteristics of interaction of the light with the sensorlayer and the scanned Fabry-Perot interferometer is registered in thesaid distribution. Judgment about the binding to be detected is madefrom a change of position of the said, at least one, maximum withrespect to values of base of the scanned Fabry-Perot interferometer.

Besides, the scanned Fabry-Perot interferometer is placed on the pathwayof the light, in which the said signal is registered, before itsincidence onto the sensor layer.

Besides, the scanned Fabry-Perot interferometer is placed on the pathwayof the said reflected or transmitted light, in which the said signal isregistered, after reflection from the sensor layer or transmissionthrough the sensor layer, respectively.

Besides, the scanned Fabry-Perot interferometer is used in thereflection mode.

Besides, the scanned Fabry-Perot interferometer is used in thetransmission mode.

Besides, the sensor layer is placed on a substrate temporarily whileregistration of the said signal or permanently.

Besides, the sensor layer is irradiated by light from the substrate'sside, the substrate being transparent, at least, partially, for thislight.

Besides, a plate with surfaces not adjacent to any substrate is used asthe sensor layer.

Besides, the liquid under test that contains or presumably contains abiological or chemical component, whose binding is the object ofdetection, is placed on one of the irradiated boundary surfaces of thesensor layer; the other boundary surface is formed with using of amaterial that provides closeness of the reflection coefficients of bothboundary surfaces; liquid with the refractive index close to therefractive index of the liquid under test is arranged on the othersurface.

Besides, liquid is placed on both irradiated boundary surfaces of thesensor layer; the liquid under test that contains or presumably containsa biological or chemical component, whose binding is the object ofdetection, being placed on at least one of these boundary surfaces; theother boundary surface is formed with using of a material that providescloseness of the reflection coefficients of both boundary surfaces.

Besides, the liquid under test is placed on both said boundary surfaces,and the said binding from the side of both said boundary surfaces isdetected.

Besides, a layer of the liquid under test that contains or presumablycontains a biological or chemical component, whose binding is the objectof detection, is used as the sensor layer. The irradiated boundarysurfaces of this layer are formed with using of hard optical materials;binding of the said component to at least one of the said boundarysurfaces is detected.

Besides, binding of at least one material component is detected inseveral spatially separated areas of the irradiated region of the sensorlayer; the said distribution is registered for each said area and usedas the said signal for each said area.

Besides, the said distribution is registered for each said area by usingsequentially in time of different values of base of the scannedFabry-Perot interferometer and measuring at each said value of intensityof the said reflected or transmitted light, summed up over the saidspectral interval, for each said area.

Besides, all the said areas are irradiated simultaneously.

Besides, several different substances capable to selectively binddiverse material components are placed in the said areas.

Besides, binding of diverse material components to the said differentsubstances is detected.

Let us explain the second variant of the method and demonstrate that itis its distinctive features that ensure the required technical result.

In this variant of the method, independence of obtained results onuncontrollable variations of intensity of the light being analyzed isprovided by placing a scanned Fabry-Perot interferometer along with thesensor layer on pathway of the light from the source to the detector.The base of the said interferometer is modulated. The distribution ofthe intensity of the said reflected or transmitted light, summed up overthe said spectral interval, as a function of base of the scannedFabry-Perot interferometer, is registered as the said signal. While thistakes place, the said base is chosen sufficiently large so that theperiod of the transmission spectrum of the scanned Fabry-Perotinterferometer is at least twice as small as the width of spectralinterval of the wavelengths participating in formation of the registeredsignal (claim 20). With regard to a Fabry-Perot interferometer, the term“base” means the length of optical path between mirrors. The mentionedcondition implied on base of the scanned Fabry-Perot interferometermeans that the spectral characteristic of transmission (reflection) ofthe interferometer contains at least two maximums. Accordingly, theFabry-Perot interferometer does not serve for extraction ofmonochromatic light, i.e. it is not a spectrum analyzer. Unlike thefirst variant of the method and the method-analogue, this variant doesnot include registration of the spectrum of the light reflected from thesensor layer or transmitted through the sensor layer.

In this variant of the method, another principle is used instead of thespectrum registration. The scanned Fabry-Perot interferometer and thesensor layer are arranged on the pathway of light participating information of the signal to be registered, the said interferometer beingplaced on the light pathway before the sensor layer or vise versa.Frequency spectrum of transmission (reflection) of the Fabry-Perotinterferometer represents a periodic function, whose maximums (minimums)are separated by frequency intervalsΔν=c/2L  (3),

where L is interferometer base. Consecutive interaction of light withthe sensor layer and the Fabry-Perot interferometer results insuperposition of spectral characteristics of these interactions.Therefore, intensity of output light summed up over all used wavelengthsis governed by correlation between spectral characteristics ofinteraction of the light with the Fabry-Perot interferometer and thesensor layer. The latter characteristic, as was discussed above, is dueto interference on the sensor layer, its frequency period is describedby formula (1), which is similar to formula (3). It follows from thestated above that the correlation signal depends upon the value of theinterferometer base. It is the dependence, which is registered in thisvariant of the method, when the interferometer base is modulated. Itshould be noted that the dependence of the intensity of the light isregistered upon a relative, not the absolute, value of theinterferometer base, i.e. upon variation of the base with respect tosome initial or mean value. If the spectral position of at least somemaximums of the characteristic of interaction of the light with theFabry-Perot interferometer and the characteristic of interaction of thelight with the sensor layer coincide, a maximum of the correlationsignal is observed in the registered dependence of the light intensityupon the interferometer base. If the optical thickness of the sensorlayer equals the interferometer base, an absolute correlation maximum isobserved. The wider the frequency spectrum of the light affecting thesensor layer is and the more frequency periods of interference on thesensor layer and frequency periods of the characteristic of theFabry-Perot interferometer fit into the spectrum width, the morepronounced the absolute correlation maximum among the other correlationmaximums is.

It is important to mention that in the proposed variant of the methodthe light beams that interact with the sensor layer and the Fabry-Perotinterferometer and contribute into the recorded signal should besufficiently collimated. First, for various beams and all wavelengths,which contribute into the recorded signal, the optical path of the lightinside the sensor layer should differ by not more than approximately afourth of wavelength. Second, the beams' directions should be within oneangular transmission maximum of the Fabry-Perot interferometer. Inmulti-channel modifications of the method, such collimation should beprovided separately in each channel or each area of the sensor layer,which is analyzed independently.

The correlation signal that governs intensity of the output lightdepends not only upon base of the Fabry-Perot interferometer, but alsoupon optical thickness of the sensor layer. The plotted dependence ofthe correlation signal on the interferometer base shifts along the axisof the interferometer base values, with a change of the said thickness.In particular, the values of the base, at which maximums of thecorrelation signal are observed, change. In this variant of the method,judgment about the binding, which is the subject of investigation (theobject of detection), is made from analysis of changes of the intensitydistribution of the output light over the interferometer base, which aredue to the said binding.

Thus, in this variant of the method the information signal is formedbased on registration of position of the correlation dependence (as arule, position of correlation maximums) with respect to the axis ofvalues of the interferometer base. Hence, in contrast to the analogues[DE 42 00 088 C2, Int. Cl. G01N 21/45, 1997 and U.S. Pat. No. 5,999,262,Int. Cl. G01B 9/02, U.S. Cl. 356/357, 1999], variations of intensity ofboth analyzed light as a whole and parts of the recorded spectrum do notaffect the information signal, which provides the said technical result.

The required technical result is achieved also in respect ofsimplification of the method, diminishing the number of necessaryoperations, decreasing of labor input and cost. Similarly to thedescribed earlier variant of the proposed method and unlike theanalogues [DE 42 00 088 C2, Int. Cl. G01N 21/45, 1997 and U.S. Pat. No.5,999,262, Int. Cl. G01B 9/02, U.S. Cl. 356/357, 1999], this variantdoes not require analytical fitting of regions of spectral dependencesand obtaining information about absolute value of the sensor layerthickness, because it uses relative measurements. It is a much simpleroperation, which concurrently provides much higher precision.

To realize this variant of the proposed method different types ofscanned Fabry-Perot interferometers known from the state of the art canbe used. In particular, the preferable types are: a) Fabry-Perotinterferometers being scanned due to a change of refraction index of amedium between mirrors, made most often on the basis of liquid crystals;b) Fabry-Perot interferometers being scanned due to a change ofinter-mirror distance by piezoelectric drivers; c) scanned Fabry-Perotinterferometers, in which the inter-mirror distance is changed byelectrostatic forces. The scanning frequency of Fabry-Perotinterferometers known from the state of the art can be chosen ratherhigh (for some types up to tens kHz) so that the scanning time and, ifnecessary, signal acquisition interval is much smaller than thecharacteristic time of the binding to be detected (as a rule, bindingtime is of order of minutes). This provides possibility to registerbinding of components in real-time.

It should be noted that this variant of the proposed method can berealized using polychromatic light as well as monochromatic light of awavelength being tuned within a chosen spectral interval. For example,one can register a correlation signal that is averaged (summed up) overseveral scanning periods over wavelength, particularly, when the time ofscanning over wavelength is significantly less than the time of scanningover the interferometer base. However, the discussed variant of themethod is realized in the simplest way and the required technical resultis achieved to the maximal degree, when polychromatic light withcontinuous spectrum is used as the light that irradiated the sensorlayer, the coherence length of the said polychromatic light and the saidreflected or transmitted light being less than double base of thescanned Fabry-Perot interferometer (claim 21). Using polychromatic lightwith continuous spectrum provides the simplest extraction of theinformation signal and diminishes the number of necessary operations.The mentioned condition for coherence length means that there are atleast two periods of the characteristic of the Fabry-Perotinterferometer within the width of spectrum of irradiating light and ofthe reflected or transmitted light being registered (see above). Toobtain a pronounced correlation signal it is important also that thereare not less than two periods of the spectral distribution due tointerference on the sensor layer within the same spectral width. Forthis, polychromatic light with coherence length that is less than thedouble thickness of the sensor layer is used (claim 22).

In the discussed here second variant of the method, information aboutbinding being detected and correspondent change of optical thickness ofthe sensor layer can be obtained from arbitrary changes of distributionof intensity of the output light over values of the scanned Fabry-Perotinterferometer base. In particular, the changes can be registered on aslope or in a minimum of this distribution. However, one can provideindependence on variations of the light intensity in the simplest wayand achieve the required technical result to the maximal extent if atleast one intensity maximum due to correlation between spectralcharacteristics of interaction of light with the sensor layer and thescanned Fabry-Perot interferometer is registered in the saiddistribution; and information about binding being detected is obtainedfrom a change of position of the said at least one maximum with respectto values of base of the scanned Fabry-Perot interferometer (claim 23).This can be an absolute correlation maximum at coincidence of the baseand optical thickness of the sensor layer as well as any side maximum.One can also observe a number of correlation maximums in the saiddistribution and register a displacement of the said distribution as awhole along values of the interferometer base.

Depending on a particular application of the method, the scannedFabry-Perot interferometer is placed on the pathway of light, in whichthe said signal is registered, before its incidence onto the sensorlayer (claim 24) or on the pathway of the said reflected or transmittedlight, in which the said signal is registered, after its reflection fromthe sensor layer or transmission through the sensor layer, respectively(claim 25). In particular, the method according to claim 24 ispreferable when it is necessary to irradiate the sensor layer by a widebeam. Then it is reasonable to send a narrow light beam to theFabry-Perot interferometer, which makes the requirements of parallelismof the interferometer mirrors and their out-of-flat displacement lessstrict, and then widen the beam to irradiate the sensor layer. Thisapproach is especially preferable in multi-channel realizations of themethod.

To achieve the required technical result, depending also on particularapplications of the method, used optical schemes, characteristics ofcomponents and operational modes of these schemes, it is reasonable touse the scanned Fabry-Perot interferometer either in the reflection mode(claim 26) or transmission mode (claim 27).

Additional features of the second variant of the method according toclaims 28-34 coincide with the additional features of the first variantof the method according to claims 6-12, which were discussed above andhave the same meaning for achievement of the technical result. For thisreason, these claims are not discussed here.

In multi-channel modifications of the second variant of the method,binding of at least one material component is detected in severalspatially separated areas of the irradiated region on the sensor layer,the said distribution is registered for each said area and the saidspectrum is used as the said signal for each said area (claim 35). Themethod according to claim 35 is similar to the method according to claim13 with the only difference, namely, the distribution of intensity ofthe said reflected or transmitted light, summed up over the usedspectral interval, as a function of base of the scanned Fabry-Perotinterferometer is registered for each said area instead of the spectrum.With this consideration, the above discussion of claim 13 is valid forclaim 35, so the latter is not discussed here.

The preferable realization of the multi-channel method consists inregistration of the said distribution for each said area by usingsequentially in time different values of base of the scanned Fabry-Perotinterferometer and measuring at each said value the intensity of thesaid reflected or transmitted light, summed up over the said spectralinterval, for each said area (claim 36). This allows recording of thesaid distribution point-by-point in parallel for all said areas of thesensor layer, each point corresponding a single value of theinterferometer base. In this case, it is sufficiently to use only onescanned Fabry-Perot interferometer to study binding of components in alarge number of areas of the sensor layer. This simplifies the method,makes it more cost-efficient and provides the required technical result.The simplest multi-channel modification is realized when all the areasare simultaneously irradiated by light (claim 37). For example, theinterferometer is located before the sensor layer on the pathway of theparallel light beam; the beam is widened after the interferometer, e.g.by a telescopic scheme; the resulting wide parallel beam is used forirradiating of all said areas. It is the simplest realization of theproposed variant of the method, which provides maximal accuracy andreliability of measurement results, and, consequently, the requiredtechnical result.

Additional features of the second variant of the method according toclaims 38 and 39 coincide with features according to claims 18 and 19 ofthe first variant of the method discussed above and are not consideredhere.

Thus, we showed that the required technical result is actually realizeddue to essential differences of the proposed variants of the method.

The third variant of the method of optical detection of binding of atleast one material component to a substance located on a surface of orinside a sensor layer due to a biological, chemical or physicalinteraction is proposed, which comprises:

-   -   irradiation of the sensor layer by light of various wavelengths,        for which the sensor layer is transparent, at least, partially;    -   registration in the reflected or transmitted light of a signal,        which depends upon optical thickness of the said sensor layer        and is due to the fact that interference on the said sensor        layer modulates the said reflection or transmission spectrum of        the said sensor layer, respectively;    -   judging about the binding being detected from a change of the        said signal,        -   which is similar to the method-analogue.

The proposed variant of the method is characterized in that:

-   -   on the pathway of the light from the source to the detector a        scanned interferometer is placed along with the sensor layer;    -   the path difference of beams of the said scanned interferometer        is chosen sufficiently large so that the period of the        transmission spectrum of the said scanned interferometer is at        least twice as small as the width of spectral range of        wavelengths participating in formation of the recorded signal;    -   the path difference of beams of the said scanned interferometer        is modulated;    -   distribution of intensity of the said reflected or transmitted        light, summed up over the said spectral interval, as a function        of the path difference of beams of the said scanned        interferometer is registered as the said signal.

This variant of the method is similar to the second variant of themethod due to employment of other interferometers, e.g. Mickelson,Mach-Zahnder interferometers or other interferometers, includingfiber-optical ones, without any change of technical entity. The saidadditional features of the second variant of the method are applicablefor the third variant of the method according to claim 97. However, inthis case, scanning of the interferometer is done by chainging the pathdifference of interfering beams (or arms) of the interferometers.

The experiments showed feasibility of the proposed variants of themethod.

To achieve the said technical result and to realize the first variant ofthe proposed method an apparatus (first variant) is proposed for opticaldetection of binding of at least one material component to a substancelocated on a surface of or inside a sensor layer due to a biological,chemical or physical interaction, which comprises:

-   -   a sensor layer;    -   a source of light, which irradiates the sensor layer, of        wavelengths that include at least operating wavelengths, for        which the sensor layer is transparent, at least, partially;    -   a detector, which is placed on the pathway of the light        reflected from the sensor layer or transmitted through the        sensor layer, for measuring the light intensity of operational        wavelengths in the spectrum of the received light;    -   a block of result generation, for example, a computer, to        generate information about the binding being detected on the        basis of changes of the said spectrum, whose input is connected        to the output of the detector,        -   which coincide with essential features of the            apparatus-analogue.

The proposed apparatus is characterized in that:

-   -   the thickness of the said sensor layer is more than 10 μm and,        at the same time, exceeds the maximal operating wavelength by at        least one order of magnitude;    -   either the source radiates collimated light or a means of        collimating the light is introduced on the pathway of the light        from the source before the sensor layer so that the sensor layer        is irradiated by collimated light.

Besides, the source is monochromatic with tunable wavelength, and thedetector is a photodetector.

Besides, the source is a tunable laser, e.g. tunable semiconductorlaser.

Besides, the source is made of a source of polychromatic light combinedwith a tunable monochromator or tunable spectral filter.

Besides, the source is a set of monochromatic sources of differentwavelengths, made with capability to switch them successively in time.

Besides, the source is a source of polychromatic light.

Besides, polychromatic light of the source has continuous spectrum withcoherence length that is less than double thickness of the sensor layer.

Besides, the detector is a matrix spectrometer.

Besides, the detector is made of a photodetector combined with a tunablemonochromator or tunable spectral filter.

Besides, the source is made based on a lamp.

Besides, the source is made based on a light-emitting diode orsuperluminescent laser diode.

Besides, the sensor layer is located on a substrate.

Besides, light from the source irradiates the sensor layer from thesubstrate's side, the substrate being transparent, at least partially,for the said light.

Besides, the sensor layer is formed by a plate with surfacesnot-adjacent to any substrate.

Besides, one of the boundary surfaces of the sensor layer, toward whichthe light from the source is directed, contacts the liquid under test,which contains or presumably contains a biological or chemicalcomponent, whose binding is the object of detection; a substance foramplification or attenuation of reflection is present on anotherboundary surface, both boundary surfaces having close reflectioncoefficients.

Besides, both boundary surfaces of the sensor layer, toward which lightfrom the source is directed, contact liquids; at least one of theseliquids being the liquid under test, which contains or presumablycontains the biological or chemical component, whose binding is theobject of detection; the refractive index of the second liquid is closeto the refractive index of the liquid under test.

Besides, both said boundary surfaces contact the liquid under test, asubstance capable to bind the said biological or chemical componentbeing present on both said boundary surfaces.

Besides, the sensor layer is formed by a layer of the liquid under test,which contains or presumably contains the biological or chemicalcomponent, whose binding is the object of detection; the boundarysurfaces of the said layer, which are irradiated by light from thesource, are formed with using of hard optical materials.

Besides, variation of optical thickness of the sensor layer within theirradiation spot of light that is directed from the source toward thesensor layer and further to the detector, does not exceed a fourth ofwavelength for the least operating wavelength.

Besides, the sensor layer consists of several spatially separated areasor comprises several spatially separated areas, which are irradiated bylight from the source, no area overlapping light incident to anotherarea; the detector is made capable to measure intensity of receivedlight for each said area; the block of result generation is made capableto analyze changes of the said spectrum for each said area and generateinformation about binding being detected based on such changes.

Besides, the sensor layer in each of the said areas is formed by aplate, which shapes the bottom of a reaction cell and is not adjacent toany substrate; the reaction cells produce an array, e.g. a microtiterplate.

Besides, the sensor layer forms the bottom of a reaction cell in eachsaid area and is located on a substrate that is transparent foroperating wavelengths; the reaction cells produce an array, e.g. amicrotiter plate.

Besides, variation of optical thickness of the sensor layer within eachsaid area does not exceed a fourth of wavelength for the least operatingwavelength.

Besides, the source is polychromatic and the detector is a set of matrixspectrometers for registration of the spectrum of received light foreach said area.

Besides, the said matrix spectrometers are furnished by optical fibersfor input of light from the said areas.

Besides, an optical system to project light from the said areas to thedetector is located on the pathway of light before the detector; thedetector comprises a set of photodetecting zones, each zone havingindependent output connected with the block of result generation.

Besides, the said optical system is made on the basis of a parabolicmirror.

Besides, the proposed apparatus differs in that:

-   -   the source is monochromatic with tunable wavelength;    -   in each said area the sensor layer is formed by a plate with        surfaces not adjacent to any substrate;    -   a control link is introduced between the block of result        generation and the source to switch the latter to another        wavelength of irradiated light after measurement by the detector        of the intensity of the received light for each said area at one        wavelength;    -   the block of result generation is made capable to generate a        spectral distribution of intensity of light measured by a        detector over wavelength for each said area.

Besides, the proposed apparatus differs in that:

-   -   the source is monochromatic with tunable wavelength;    -   in each said area the sensor layer is formed by a layer of the        liquid under test, which contains or presumably contains the        biological or chemical component, whose binding is the object of        detection; the boundary surfaces of the said layer, which are        irradiated by light from the source, are formed with using of        hard optical materials;    -   a control link is introduced between the block of result        generation and the source to switch the latter to another        wavelength of irradiated light after measurement by the detector        of the intensity of the received light for each said area at one        wavelength;    -   the block of result generation is made capable to generate a        spectral distribution of intensity of light measured by the        detector over wavelength for each said area.

Besides, the proposed apparatus is characterized in that:

-   -   the source is polychromatic;    -   there is a tunable monochromator or tunable spectral filter        placed on the light pathway before the detector;    -   a control link is introduced between the block of result        generation and the said tunable monochromator or tunable        spectral filter to switch the latter to another wavelength after        measurement by the detector of the intensity of the received        light for each said area at one wavelength;    -   the block of result generation is made capable to generate a        spectral distribution of intensity of light measured by the        detector over wavelength for each said area.

Besides, different substances capable to selectively bind differentmaterial components from a gaseous or liquid medium are located in thesaid areas.

As a realization of the second variant of the proposed method the secondvariant of the apparatus is proposed, which is intended for opticaldetection of binding of at least one material component to a substancelocated on a surface of or inside the sensor layer due to a biological,chemical or physical interaction. The apparatus comprises:

-   -   a sensor layer;    -   a source of light, which irradiates the sensor layer, of        wavelengths that include at least operating wavelengths, for        which the sensor layer is transparent, at least, partially;    -   a detector, which is placed on the pathway of the light        reflected from the sensor layer or transmitted through the        sensor layer, for measuring intensity of light of operating        wavelengths;    -   a block of result generation, for example, a computer, to        generate information about the binding being detected, whose        input is connected to the output of the detector,        -   which coincide with essential features of the            apparatus-analogue.

The proposed apparatus is characterized in that:

-   -   on the pathway of the light from the source to the detector a        scanned Fabry-Perot interferometer with modulated base (i.e.        length of optical path between the mirrors) is placed along with        the sensor layer;    -   the base of the scanned Fabry-Perot interferometer is        sufficiently large so that the related period of the        transmission spectrum of the said scanned Fabry-Perot        interferometer is at least twice as small as the width of        spectral range, corresponding to operating wavelengths;    -   block of signal generation is made capable to register        distribution of the light intensity measured by the detector,        summed up over the operating wavelengths, as a function of base        of the scanned Fabry-Perot interferometer and generate        information about the binding being detected on the basis of a        change of the said distribution.

Besides, light from the source, which irradiates the sensor layer andthe scanned Fabry-Perot interferometer, is collimated, i.e. a source ofcollimated light is used as the light source and/or means forcollimation of the light are introduced before the sensor layer and/orthe scanned Fabry-Perot interferometer;

Besides, the source is a source of polychromatic light with continuousspectrum and coherence length that is less than the double base of thescanned Fabry-Perot interferometer.

Besides, the coherence length of the said polychromatic light is lessthan the double thickness of the sensor layer.

Besides, the source is made on the basis of a lamp.

Besides, the source is made on the basis of a light-emitting diode orsuperluminescent laser diode.

Besides, the scanned Fabry-Perot interferometer is introduced in theoptical scheme as a reflecting element.

Besides, the scanned Fabry-Perot interferometer is introduced in theoptical scheme as a transmitting element.

Besides, the scanned Fabry-Perot interferometer is located on thepathway of light from the source to the sensor layer.

Besides, at least part of the path from the source to the scannedFabry-Perot interferometer and/or from the scanned Fabry-Perotinterferometer to the sensor layer the light travels inside an opticalfiber.

Besides, the scanned Fabry-Perot interferometer is located on thepathway of light from the sensor layer to the detector.

Besides, the sensor layer is located on a substrate.

Besides, light from the source irradiates the sensor layer from thesubstrate's side, the substrate being transparent, at least, partially,for the light.

Besides, the sensor layer is formed by a plate with surfaces notadjacent to any substrate.

Besides, one of the boundary surfaces of the sensor layer, towards whichthe light from the source is directed, contacts the liquid under test,which contains or presumably contains the biological or chemicalcomponent, whose binding is the object of detection; a substance foramplification or attenuation of reflection is present on anotherboundary surface, both boundary surfaces having close reflectioncoefficients.

Besides, both boundary surfaces of the sensor layer, toward which lightfrom the source is directed, contact liquids, at least one of theseliquids being the liquid under test, which contains or presumablycontains the biological or chemical component, whose binding is theobject of detection.

Besides, both said boundary surfaces contact the liquid under test, asubstance capable to bind the said biological or chemical componentbeing present on both said boundary surfaces.

Besides, the sensor layer is formed by a layer of the liquid under test,which contains or presumably contains the biological or chemicalcomponent, whose binding is the object of detection; the boundarysurfaces of the said layer, which are irradiated by light from thesource, are formed with using of hard optical materials.

Besides, variation of optical thickness of the sensor layer within theirradiation spot by light that is directed from the source toward thesensor layer and further to the detector, does not exceed a fourth ofwavelength for the least operating wavelength.

Besides, the sensor layer consists of several spatially separated areasor comprises several spatially separated areas, which are irradiated bylight from the source, no area overlapping light incident to anotherarea; the detector is made capable to measure intensity of the receivedlight for each said area; the block of result generation is made capableto analyze changes of the said distribution for each said area andgenerate information about the binding being detected based on suchchanges.

Besides, the sensor layer in each said area forms the bottom of areaction cell, and the reaction cells produce an array, e.g. in the formof a microtiter plate.

Besides, variation of optical thickness of the sensor layer within eachsaid area does not exceed a fourth of wavelength for the least operatingwavelength.

Besides, an optical system to project light from the said areas to thedetector is arranged on the pathway of light before the detector; thedetector comprises a set of photodetection zones, each zone havingindependent output connected with the block of result generation.

Besides, the said optical system is made on the basis of a parabolicmirror.

Besides, the proposed apparatus is characterized in that:

-   -   the source is a source of polychromatic light with continuous        spectrum and coherence length that is less than the double base        of the scanned Fabry-Perot;    -   a control link is introduced between the block of result        generation and the scanned Fabry-Perot interferometer to switch        the latter to another value of the base after measurement by the        detector of the intensity of the received light for each said        area at one value of the base;    -   the block of signal generation is made capable to obtain        distribution of intensity of the light measured by the detector,        summed up over the operating wavelengths, at various values of        the scanned Fabry-Perot base for each said area.

Besides, different substances capable to selectively bind differentmaterial components from a gaseous or liquid medium are arranged in thesaid areas.

Besides, an optical system for projection of image of the sensor layeronto the detector is arranged on the pathway of light before thedetector; the detector comprises a set of photodetector zones; each zonehas an independent output connected with the block of signal generation.

As a realization of the third variant of the proposed method the thirdvariant of the apparatus is proposed, which is intended for opticaldetection of binding of at least one material component to a substancelocated on a surface of or inside the sensor layer due to a biological,chemical or physical interaction. The apparatus comprises:

-   -   a sensor layer;    -   a source of light, which irradiates the sensor layer, of        wavelengths that include at least operating wavelengths, for        which the sensor layer is transparent, at least, partially;    -   a detector, which is placed on the pathway of the light        reflected from the sensor layer or transmitted through the        sensor layer, for measuring intensity of light of operating        wavelengths;    -   a block of result generation, for example, a computer, to        generate information about the binding being detected, whose        input is connected to the output of the detector,        -   which coincide with essential features of the            apparatus-analogue.

The proposed apparatus is characterized in that:

-   -   on the pathway of the light from the source to the detector a        scanned interferometer with modulated path difference of beams        is placed along with the sensor layer;    -   the path difference of beams of the said scanned interferometer        is sufficiently large so that the related period of the        transmission spectrum of the said scanned interferometer is at        least twice as small as the width of spectral range,        corresponding to operating wavelengths;    -   block of signal generation is made capable to register        distribution of intensity of the light measured by the detector,        summed up over the operating wavelengths, as a function of path        difference of beams of the scanned interferometer and generate        information about the binding being detected on the basis of a        change of the said distribution.

Besides, additional features of the second variant of the apparatusextend to the third variant of the apparatus. However, in this case,scanning of the interferometer is done by changing the path differenceof interfering beams (or arms) of the interferometer.

BRIEF DESCRIPTION OF FIGURES

FIG. 1. A scheme of first variant of the proposed apparatus, claim 40.

FIG. 2. A scheme of second variant of the proposed apparatus, claim 71.

The following notations are used in FIG. 1-2: 1—source; 2—means forcollimation of light; 2.1 and 2.2 are its components; 3—light-splittingelement; 4—sensor layer; 5—optical system for projection of light ontodetector; 6—detector; 7—block of signal generation; 8—scannedFabry-Perot interferometer.

MODES FOR CARRYING OUT THE INVENTION

First variant of the proposed apparatus is schematically shown inFIG. 1. A substance (or several substances) capable to bind material(biological, chemical or other) components due to a biological, chemicalor physical interaction is located on a surface of or inside a sensorlayer 4. The substance contacts a medium (not shown in FIG. 1) thatcontains or presumably contains the said components. Biospecific binding(e.g. antigen-antibody, biotin-avidin), formation of chemicalassociations, adsorption or absorption, etc. can be named among examplesof such interactions. For operation of the apparatus it is imperativenot the type of interaction, but the fact that any such binding goeswith superinduction of a new substance to the surface of or into thesensor layer and causes an increase (a decrease in variants according toclaims 57 and 68, when the sensor layer is formed by the medium undertest) of optical thickness of the sensor layer, which is registered bythe apparatus. The apparatus can register also reverse reactions ofdetachment of components from the substance of the sensor layer, becausethey go with a decrease (an increase in claims 57 and 68) of opticalthickness of the sensor layer.

The sensor layer 4 is irradiated by light from a source 1, whichcontains simultaneously or separately different operating wavelengths.The operating wavelengths are those of light from the source 1, whoseintensity is measured by a detector 6. In general case, there can bepresent also wavelengths in the spectrum of the source 1 (e.g.incandescent lamp), which are beyond the range of sensitivity of thedetector 6 (e.g. produced on the basis of semiconductor photodiodes).The sensor layer 4 is transparent for operating wavelengths to thedegree, which provides a possibility to observe interference ofsecondary light waves that arise on boundary surfaces of the sensorlayer 4.

The detector 6 is located on the pathway of light reflected from thesensor layer 4 (this variant of the apparatus is shown in FIG. 1) ortransmitted through the sensor layer 4 (this variant is not shown). Thedifference between the apparatus variants, which use measurements in thereflected and transmitted light, is insignificant, and both variantsprovide the same technical result. A light-splitting element 3 in thescheme shown in FIG. 1 is one of the said insignificant differences. Onecan employ, for example, a light-splitting plate (shown in FIG. 1) or alight-splitting cube as the element 3. It is reasonable to use theelement 3 for splitting beams of light, which is incident to the sensorlayer and reflected from it, in the schemes of the apparatus employedfor measurements in the reflected light and the angle of the lightincidence onto the sensor layer is close to the normal. In measurementsin the transmitted and reflected light at large angles of incidence withrespect to the normal to the sensor layer the element 3 is notnecessary.

The optical system 5 for projection of light from the sensor layer tothe detector is not a necessary element in the apparatus according toclaim 40. It is reasonable to employ the system 5 when, for example,there is a substantial difference in size of the irradiated region ofthe sensor layer 4 and photodetecting zone of the photodetector 6, toprevent significant losses of light and to increase signal-to-noiseratio. The system 5 is used also in a number of multi-channelmodifications of the apparatus (see below). Lens or mirror objectives(e.g. with using of a parabolic mirror), telescopic systems, etc. canserve as the system 5.

Operation of the first variant of the apparatus is based on the factthat the detector 6 registers spectrum of the received light bymeasuring intensity of the light simultaneously or separately atdifferent operating wavelengths. An interference pattern, which isperiodic over light frequency with the period described by therelationship (1), is observed in the registered spectrum. A block ofsignal generation 7 analyzes changes of the said interference pattern,which is due to a change of optical thickness of the sensor layer 4during binding being detected, and generates information about thebinding being detected.

As was mentioned above during analysis of the method, the light beamshould be sufficiently collimated to observe the said interferencepattern. If the source 1 itself does not provide sufficient collimationof the output light beam (e.g. if the source is a lamp, light-emittingdiode or a semiconductor laser), a means 2 for collimating light, whichdecreases beam divergence, is introduced on the pathway of the lightfrom the source 1 before the sensor layer 4. A collimating objective(lens or mirror), aperture, combination of an objective with anaperture, etc. can serve as examples of the means 2. The means 2 is notemployed, if the source 1 is a source of collimated light, e.g. a laserwith low divergent output beam.

Contrasting to the apparatus-analogue, in the proposed apparatus largethickness of the sensor layer 4 leads to presence of severalinterference periods in the recorded spectrum. Therefore, it is notnecessary to measure intensity precisely and determine on that basis anabsolute thickness of the sensor layer. Instead, a spectral shift ofindividual interference maximums or minimums, or of whole “comb” ofmaximums or minimums is analyzed, which serves as a source ofinformation about relative changes of optical thickness of the sensorlayer 4 and, consequently, about the binding being detected.

Since the spectral position of interference maximums and minimums doesnot depend upon variations of intensity of the whole spectrum or itsparts, the proposed variant of the apparatus provides the requiredtechnical result. The more detailed discussion is provided above whileanalysis of the first variant of the proposed method.

To achieve the required technical result in part of increasing accuracyof measurements with simultaneous decrease of requirements to thedetector 6, it is reasonable to implement the scheme of the apparatus,in which the source 1 is monochromatic with tunable wavelength and thedetector 6 is a photodetector (claim 41). Preferable realizations arethose, in which the source 1 is a tunable laser, e.g. a tunablesemiconductor laser (claim 42); or the source 1 is made of a source ofpolychromatic light in combination with a tunable monochromator ortunable spectral filter (claim 43); or the source 1 is a set ofmonochromatic light sources of different wavelengths, which is madecapable to switch the wavelengths successively in time (claim 44). Suchvariants of the apparatus are especially preferable for purposes ofmulti-channel registration.

It should be emphasized that a qualitative difference of the firstvariant of the proposed apparatus and related method from the analogue[DE 42 00 088 C2, Int. Cl. G01N 21/45, 1997] is in capability of usingtunable lasers as the light source. The tuning range of such lasers iscommonly quite narrow, so in the analogue it could only be a small partof width of the spectrum necessary to find optical thickness of thesensor layer and detect binding of components. In the proposed methodand apparatus, using of large thickness of the sensor layer providespresence of several interference periods within tuning range of thelaser, which is sufficient for high precision detection of binding ofcomponents. Thus, in contrast to the analogue, in the proposed methodand apparatus it is possible to use tunable lasers and othermonochromatic sources tunable in narrow spectral ranges, and alsopolychromatic sources with narrow spectral range, e.g. light-emittingdiodes or superluminescent laser diodes (see below).

Extra opportunities are open, if the source 1 is a source ofpolychromatic light (claim 45). Requirements to the source are weaker,and the number of operations is smaller, which makes the apparatussimpler and more cost-efficient, especially at registration on onechannel or small number of channels. In this case, it is reasonable touse light with continuous spectrum and coherence length that is lessthan double thickness of the sensor layer 4 (claim 46). Continuity ofthe spectrum permits one to obtain the spectral position of interferencemaximums or minimums with maximal precision. The mentioned condition forcoherence length means that there are at least two interference maximumsor minimums of intensity of the transmitted or reflected light withinspectral width of the used polychromatic light.

The apparatus with using of polychromatic light, in which the detector 6is a matrix spectrometer (claim 47) is especially preferable forsimplicity of realization, reproducibility of results and diminishing ofthe number of necessary operations. As a rule, the matrix spectrometeris made based on a dispersion element, e.g. a diffraction grating,combined with a matrix photodetector such as an array of photodiodes ora CCD-array. Usually, a linear (one-dimensional) array (matrix) orientedalong the dispersion direction is used. Light is supplied into suchspectrometer through an aperture (slot) or an optical fiber. The matrixspectrometer permits one to register simultaneously (taking into accountacquisition time) the whole required spectrum and avoid scanning overthe spectrum. However, depending on particular applications, thedetector 6, which is made based on a photodetector combined with atunable monochromator or tunable spectral filter, can be used incombination with a source of polychromatic light (claim 48).

It is reasonable to make the source of polychromatic light based on alamp (claim 49) or a light-emitting diode (claim 50). In the first case,there is a widest spectrum of the source, which provides versatility forregistration of the spectrum in various ranges of wavelengths, andchoice of a desirable and rather large number of interference periodswithin the width of the recorded spectrum. In the latter case, thelargest spectral density of power of the used polychromatic light isprovided, which is profitable for increasing of the signal-to-noiseratio. It should be noted that due to large thickness of the sensorlayer, several periods of interference can be registered within spectrumof a light-emitting diode, which is an important difference from theanalogue.

Features of the apparatus according to claims 51-57 were consideredabove while analysis of claims 6-12 relevant to the first variant of themethod, so the corresponding discussion is not repeated here. It shouldbe noted only that in the apparatus (claim 56) that realizes the methodfor investigation of binding of a component (components) from the sideof both boundary surfaces of the sensor layer (claim 11 of the method),there is a substance capable to bind respective component (components)on both said boundary surfaces. For example, it can be an antigen forbinding a corresponding antibody, streptavidin for binding byotinylatedproteins, nucleotide chains for binding complementary nucleotidesequences and other substances for specific binding. Non-specificbinding can be also used.

An important feature of the proposed apparatus is that a variation ofoptical thickness of the sensor layer 4 within the irradiation spot bylight directed from the source 1 to the sensor layer 4 and further tothe detector 6 does not exceed a fourth of wavelength for the leastoperating wavelength (claim 58). The said restriction on the thicknessvariation refers to the irradiation spot of the light, which contributeto generation of the information signal registered by the detector 6.Optical thickness of the sensor layer in every point of its surface isdescribed by expression (2) that takes into account a distribution ofrefraction index over the layer thickness, the refraction index and,consequently, optical thickness, being dependent on light wavelength. Atlarge variations of optical thickness over the irradiated regionmaximums and minimums of interference patterns in spectra of differentareas would overlap, there would occur a decrease of contrast, blurringor, maybe, even disappearance of the well-defined interference patternin the resulting spectrum averaged over the irradiated region. However,if these variations do not exceed a fourth of wavelength for the leastwavelength of the registered spectrum, then a distinct interferencepattern is observed over whole registered spectrum, which permits one toachieve the required technical result.

An apparatus for multi-channel registration of binding components isrealized as follows: the sensor layer 4 consists of several spatiallyseparated areas or comprises several spatially separated areas, whichare irradiated by light from the source 1; no area overlaps lightincident to another area; the detector 6 is made capable to measureintensity of the received light for each said area; the block of signalgeneration 7 is made capable to analyze changes of the said spectrum foreach said area and generate information about the binding being detectedon the basis of these changes (claim 59). Principles of multi-channelregistration, fields of application and provision of the requiredtechnical result were discussed above while analysis of the method. Theregistration channels correspond to spatially separated areas of theirradiated region or groups of the said areas of the sensor layer, andthe information signal (in the first variant of the method andapparatus—the spectrum) is recorded for each area separately andindependently of other areas. The block of signal generation generatesinformation about binding being detected for either each said area or agroup of the said areas, e.g. by averaging of information signals overthe group of areas.

In some cases, the said areas can be chosen or formed on a surface of acontinuous sensor layer (a film, plate or liquid layer), for example, bydeposition of different binding (recognition) reagents onto variousregions of the surface. If the binding being detected takes place onboth boundary surfaces of or inside the sensor layer, then regions ofthe said two surfaces or volume, which are irradiated by the same beamsfrom the source, are considered as a single area. In other cases, thesensor layer can be non-continuous and represents a set of spatiallyseparated areas, e.g. bottom plates of an arbitrary set of reactioncells.

In particular, in one preferable realization the sensor layer in eachsaid area is formed by a plate, which shapes the bottom of a reactioncell and is not adjacent to any substrate, and the reaction cellsproduce an array, e.g. a microtiter plate (claim 60). In anotherpreferable realization of the apparatus, the sensor layer in each saidarea forms the bottom of a reaction cell and is located on a substratethat is transparent for operating wavelengths, and the reaction cellsproduce an array, e.g. a microtiter plate (claim 61). In both cases,there can be either a single area of the sensor layer, a signal fromwhich is registered separately and independently from other areas, orseveral said areas within one cell. Fabrication of the base element ofthe apparatus as a microtiter plate provides compatibility with standardformats of biological and biochemical analyses, decreases cost of theapparatus and the number of necessary operations, extends the field ofapplication, providing the required technical result.

For the multi-channel apparatus, it is important that variation ofoptical thickness of the sensor layer within each said area does notexceed a fourth of wavelength for the least operating wavelength (claim62), i.e. the discussed above restriction on variation of thicknesswithin the irradiation spot should be extended to each area of thesensor layer, the signal from which is registered separately andindependently from other areas.

At a relatively small number of channels the required technical resultis provided maximally if the source is polychromatic, the detector is aset of matrix spectrometers for registration of spectrum of the receivedlight for each said area (claim 63), one area being correspondent to oneregistration channel (one reaction cell). This allows registration ofthe whole spectrum in each channel, avoiding thus scanning overspectrum. This also decreases the number of necessary operations andtime consumption, increases accuracy, reliability of results and timeresolution, which is important for real-time registration. There areextra opportunities if the said matrix spectrometers are furnished byoptical fibers to input light from the said areas (claim 64) so that thelight from the said areas is supplied to the spectrometers. This permitsone to miniaturize the said areas to the size of about the fibers'diameter, diminish weight and dimensions of the whole apparatus, makespossible remote investigations, when the detector is located at asignificant distance.

In other cases, in particular, at a large number of channels, an opticalscheme 5 for projection of light from the said areas to the detector 6is placed on the pathway of the light before the detector 6; thedetector 6 comprises a set of photodetecting zones, each zone havingindependent output connected with the block of result generation 7(claim 65). The photodetector 6 can be made as a set of photodetectors,a two-dimensional photodetector array, a CCD-array, or a camcorder. Inthese cases, an area of the sensor layer, a signal from which isregistered separately and independently from other areas, is an area ofthe sensor layer, the light from which is projected onto the samephotodetecting zone. One or several said areas (in preferable variants,a large number of the said areas and large number of photodetectingzones) correspond to one reaction cell (one registration channel). Itshould be emphasized, that the restriction discussed above (claim 62)concerning variation of optical thickness of the sensor layer refers toonly one said area, not to the whole reaction cell. Therefore, withincrease of the number of the said areas and, accordingly,photodetecting zones per one reaction cell, the requirements to evennessof thickness of the sensor layer within the reaction cell decrease. Thissignificantly reduces cost of the apparatus and contributes toachievement of the required technical result. In addition, accuracy andreliability of results of measurements increase due to averaging of thesignals from individual areas over the whole irradiated region of thesensor layer within the reaction cell.

The said optical system 5 serves usually for both deflection andfocusing of light beams, because dimensions of photodetecting zones ofthe detector 6 are typically considerably less than dimensions of areasof the sensor layer under study, for example, in the case, when thesensor layer 4 matches a microtiter plate of typical size. Such opticalsystem 5 may comprise, for example, a lens or mirror objective,telescopic system, etc. The system 5 can generate an image of the sensorlayer 4 on the detector 6. However, this is not necessary due to the useof collimated light. In preferable realization of the apparatus, thesaid system is made on the basis of a parabolic mirror (claim 66). Thisallows one to use small photodetector arrays, minimize the optical pathfrom the sensor layer to the detector, and minimize weight anddimensions of the apparatus, simultaneously avoiding aberrations. Thisincreases sensitivity of the apparatus and accuracy of measurements.

To achieve the required technical result in the apparatus according toclaim 65, it is reasonable to provide registration of the spectrummodulated by interference on the sensor layer by scanning over thespectrum, i.e. by measuring intensity in the spectrum sequentially atdifferent wavelengths. This considerably simplifies the apparatus due toreduced requirements to the multi-spot detector: it is necessary tomeasure intensity of the light incident to each spot, not to registerits spectrum. There is further potential to simplify and reduce cost ofthe apparatus in using a thick sensor layer in the form of a plate. Insuch apparatus (claim 67):

-   -   the source is monochromatic with tunable wavelength;    -   in each said area the sensor layer is formed by a plate with        surfaces not adjacent to any substrate;    -   a control link is introduced between the block of result        generation and the source to switch the latter to another        wavelength of irradiated light after measurement by the detector        of the intensity of the received light for each said area at one        wavelength;    -   the block of result generation is made capable to generate a        spectral distribution of intensity of the light measured by the        detector over wavelength for each said area.

In another version the thick sensor layer is formed by the liquid beingtested. Advantages of such scheme were discussed above. In suchapparatus (claim 68):

-   -   the source is monochromatic with tunable wavelength;    -   in each said area the sensor layer is formed by a layer of the        liquid under test, which contains or presumably contains the        biological or chemical component, whose binding is the object of        detection; the boundary surfaces of the said layer, which are        irradiated by light from the source, are formed with using of        hard optical materials;    -   a control link is introduced between the block of result        generation and the source to switch the latter to another        wavelength of irradiated light after measurement by the detector        of the intensity of the received light for each said area at one        wavelength;    -   the block of result generation is made capable to generate a        spectral distribution of intensity of the light measured by the        detector over wavelength for each said area.

Another option to scan over the spectrum is changing of wavelength ofthe radiation received by the detector, not the radiation emitted by thesource. In the corresponding apparatus (claim 69):

-   -   the source is polychromatic;    -   there is a tunable monochromator or tunable spectral filter        placed on the light pathway before the detector;    -   a control link is introduced between the block of result        generation and the said tunable monochromator or tunable        spectral filter to switch the latter to another wavelength after        measurement by the detector of the intensity of the received        light for each said area at one wavelength;    -   the block of result generation is made capable to generate        spectral distribution of intensity of the light measured by the        detector over wavelengths for each said area.

To make all the mentioned multi-channel modifications of the apparatusapplicable to simultaneous registration of binding of differentcomponents of gaseous and liquid media as well as analysis of presenceof simultaneously several components in gaseous and liquid media,providing the required technical result (see above the analysis of themethod), there are different substances, which are capable toselectively bind different material components from a gaseous and liquidmedium, arranged in the said areas (claim 70).

The second variant of the proposed apparatus (one of possible schemes)is schematically shown in FIG. 2. The sensor layer 4 is irradiated bylight from the source 1, which contains simultaneously or separatelydifferent operating wavelengths. As already discussed for the firstvariant of the apparatus, a substance that realizes the binding beingdetected, which causes a change of optical thickness of the sensorlayer, is arranged on the surface of or inside the sensor layer 4. Thesensor layer is transparent for operating wavelengths to the degree,which provides possibility to observe interference of secondary lightwaves that arise on boundary surfaces of the sensor layer. As a resultof such interference, the transmission and reflection spectra of thesensor layer are a modulated intensity distribution with a period on thelight frequency described by relationship (1). Only the scheme that useslight reflected from the sensor layer is shown in FIG. 2. The schemethat uses transmitted light differs insignificantly (in particular, asin the first variant, it does not require the light-splitting element 3)and provides the same technical result.

Spectrum of the light at the output of Fabry-Perot interferometer alsorepresents a modulated distribution of intensity with a period on thelight frequency described by relationship (3). The base of the scannedFabry-Perot interferometer 8 in the proposed apparatus is chosensufficiently large so that the period of the spectral characteristic ofthe interferometer 8 is at least twice as small as the width of spectralrange, corresponding to operating wavelengths. This means that there areat least two maximums of the spectral characteristic of theinterferometer within the width of spectrum of the operatingwavelengths.

Operation of the second variant of the apparatus is based on the factthat the interferometer 8 is scanned and its base is modulated. Undermodulation of base the period and spectral positions of maximums of thespectral characteristic of the interferometer change according to therelationship (3). Contrasting to the apparatus-analogue and firstvariant of the apparatus, in this variant, the intensity of the lightafter interaction with the sensor layer 4 and the scanned Fabry-Perotinterferometer 8 is registered instead of the spectrum. The saidintensity is measured by the detector 6, e.g. a photodetector orphotodetector array. The block of signal generation 7 registers thedependence of this intensity upon base of the interferometer 8. Whenspectral positions of at least several spectral maximums in thecharacteristics of the interferometer 8 and the sensor layer 4 coincide,a correlation maximum is observed in the said dependence; when positionsof all maximums coincide, there is an absolute correlation maximum. Whenoptical thickness of the sensor layer 4 changes as a result of thebinding being detected, the dependence of intensity upon base of theinterferometer 8 shifts along the base values, which is especiallyconvenient to observe by a shift of the correlation maximums. Analyzingthe said shift, the block of signal generation 7 generates and indicatesinformation about the binding being detected.

As was mentioned during analysis of the method, the light beams, whichinteract with the sensor layer 4 and Fabry-Perot interferometer 8 andcontribute to the recorded signal, should be sufficiently collimated.First, variations of optical path inside the sensor layer 4 fordifferent beams and operating wavelengths should not exceed about afourth of wavelength. Second, directions of the beams should be withinone angle transmission maximum of the Fabry-Perot interferometer 8.Because of this, on the pathway of light from the source a means forlight collimation can be introduced, which decreases beam divergence ifthe light from the source is not sufficiently collimated. A collimatingobjective (lens or mirror), aperture, combination of an objective withan aperture, etc. can serve as examples of the said means. If there areno objects, which change the divergence of the light beam or its size incross section, between the Fabry-Perot interferometer and the sensorlayer, then the said collimating means can be introduced before only oneof the said elements (the sensor layer or interferometer), which thelight meets first on its pathway from the source. If there are objects,which change the divergence of the beam or its size in cross section(e.g. if at least part of the path from the Fabry-Perot interferometerto the sensor layer the light travels through an optical fiber), then atleast one more means for collimation of light is introduced before thesecond element of the mentioned ones. As an example, a scheme of theapparatus is shown in FIG. 2, in which the source 1 is a source ofcollimated light incident onto the interferometer 8 (or a means forcollimation of light is embedded into the source 1), so a separate meansfor collimation of light is not provided before the first element,interferometer 8. A means for collimating light 2 is introduced beforethe second element, the sensor layer 4. The means 2 also serves fordivergence of the light beam, directed from the interferometer 8 to thesensor layer 4. It is desirable to illuminate the Fabry-Perotinterferometer 8 by a narrow light beam (this decreases requirements toflatness and parallelism of the interferometer mirrors). On thecontrary, a wide beam is desirable for illumination of the sensor layer,which increases the signal-to-noise ratio due to averaging over surfaceof the sensor layer 4. In multi-channel modifications of the apparatusthis allows one to register binding of components in a large number ofareas of the sensor layer 4 in parallel and independently. In FIG. 2 themeans 2 consists of two elements, a lens 2.1 and parabolic mirror 2.2,the mirror 2.2 simultaneously serves as the optical system 5 forprojection of the light from the sensor layer 4 to the detector 6.

Additional features of the apparatus according to claims 72, 73 coincidewith features of the method according to claims 21, 22 discussed abovewhile analysis of the method and are not considered here.

Additional features of the apparatus according to claims 74, 75 coincidewith features of the first variant of the apparatus according to claims49, 50 discussed above and are not considered here.

Additional features of the apparatus according to claims 76, 77, 78, 80coincide with features of the method according to claims 26, 27, 24, 25,respectively, discussed while analysis of the method and are notconsidered here. However, a preferable realization of the apparatusaccording to claim 78 should be mentioned, in which the light travelsinside an optical fiber at least part of its way between the source andthe scanned Fabry-Perot interferometer and/or the scanned Fabry-Perotinterferometer and the sensor layer (claim 79). This modificationprovides the required technical result due to decreasing of weight anddimensions, and increasing of functional versatility of the apparatus.In addition, the modification is important for remote measurements.

Additional features of the apparatus according to claims 81-88 coincidewith features of the first variant of the apparatus according to claims51-58 discussed above and are not considered here. Additional featuresof the apparatus according to claim 89 are similar to features of thefirst variant of the apparatus according to claim 59 with the onlydifference, namely, the distribution of intensity of light over valuesof base of the scanned Fabry-Perot interferometer is considered insteadof the spectrum. Taking into account this notice for multi-channelmodifications (claim 89) of the second variant of the apparatus, thediscussion above is valid, so claim 89 is not discussed here.

Claim 90 describes a multi-channel apparatus that comprises severalspatially separated areas of the sensor layer, where the sensor layer ineach said area forms the bottom of a reaction cell, and the reactioncells produce an array, e.g. a microtiter plate. Importance ofrealization of the base element on the basis of the microtiter plate isanalyzed above for the first variant of the apparatus (claims 60, 61).The difference of the second variant (claim 90) is in that there are norestrictions on thickness of the sensor layer due to the principle ofoperation of this variant of apparatus. Implementation of the sensorlayer on a substrate (with an arbitrary set of other layers or withoutthem) or as a separate plate for each said area is also implied.

Additional features of the apparatus according to claims 91, 92, 93coincide with features of the first variant of the apparatus accordingto claims 62, 65, 66 discussed above and are not considered here.

An apparatus according to claim 94 is proposed as a realization of themethod according to claim 36 and differs in that:

-   -   the source is a source of polychromatic light with continuous        spectrum and coherence length that is less than the double base        of the scanned Fabry-Perot interferometer;    -   a control link is introduced between the block of result        generation and the scanned Fabry-Perot interferometer to switch        the latter to another value of the base after measurement by the        detector of the intensity of the received light for each said        area at one value of the base;    -   the block of signal generation is made capable to obtain        distribution of intensity of the light measured by the detector,        summed up over the operating wavelengths, as a function of        values of the Fabry-Perot base for each said area.

As was mentioned while analysis of the method according to claim 36,this scheme allows point-by-point registration of the said distributionof intensity over values of the interferometer base simultaneously onall channels, taking one value of the interferometer base after another.In this case, one interferometer serves all channels simultaneously andprovides high accuracy of measurements. This simplifies the apparatusand provides the required technical result.

Additional feature of the apparatus according to claim 95 coincides withthe feature of the first variant of the apparatus according to claim 70discussed above and is not considered here.

Additional feature of the apparatus according to claim 96 permitsaveraging of thickness difference of biomolecular layers whilebiochemical analyses. In microbiological and bacteriological analyses,it also allows direct calculation of the quantity of bonded biologicalagents (bacteria, microorganisms, etc.) with spatial resolution alongthe sensor layer. In this case, the apparatus is highly stable totemperature or other drifts, because only registration of changes of thesensor layer contrast is important.

The third variant of the apparatus according to claim 98 is similar tothe second variant of the apparatus. However, it employs otherinterferometers, e.g. Mickelson, Mach-Zahnder interferometers or otherinterferometers, including fiber-optical ones, without any change oftechnical entity. In this case, scanning of the interferometer is doneby choice of the path difference of interfering beams (or arms) of theinterferometer. The mentioned additional features of the second variantof the apparatus are applicable to the third variant of the apparatusaccording to claim 98.

Additional features of the method and apparatus according to claims 99and 100, respectively, allow one to provide high accuracy ofmeasurements working with scanned interferometers and high spatialresolution of registration of near-surface reactions, particularly withmulti-channel implementation of the apparatus.

Thus, it has been shown that the required technical result is achieveddue to the essential differences of the proposed variants of theapparatus.

Experiments demonstrated feasibility of the proposed variants.

INDUSTRIAL APPLICABILITY

The proposed invention is intended for detection and investigation ofbinding of biological and chemical components of media to a substance ofsensor layers on the basis of a biological, chemical or physicalinteraction by registration of optical signals due to interference onthese sensor layers. The invention can be applied for real-timeregistration of the mentioned processes of binding, opposite processesof detachment, and investigation of kinetics of the processes. Byregistration of parameters of binding of biological and chemicalcomponents to sensing materials the invention permits one to determinethe content and measure concentrations of the said components indifferent media under test, mostly in biological solutions. Inparticular, the invention is applicable for immunological analyses andallows one to register in real-time the binding of antigens andantibodies, and determine their presence in biological solutions withoutusing of radioactive, ferment, fluorescent and other labels. Besides,the invention permits one to carry out parallel biochemical analyses ofa large number of probes with high throughput capacity, and alsomulti-component analyses. The invention is applicable for production of“biochips”, “gene chips”, optoelectronic “nose” and “tongue”, etc.

1. A method of optical detection of binding of at least one materialcomponent to a substance due to a biological, chemical or physicalinteraction, said method comprising the steps of: (a) providing a sensorlayer formed by a solid optical material, said sensor layer having twoboundary surfaces separated from each other by a distance of more than10 μm; (b) immobilizing said substance on at least one of said boundarysurfaces; (c) contacting a gaseous sample or liquid sample, whichcontains said material component, with the at least one of said boundarysurfaces so that interaction between the material component and thesubstance takes place; (d) irradiating said sensor layer by a beam ofpolychromatic light, comprising a spectrum with a plurality ofwavelengths, for which said solid optical material is at least partiallytransparent, wherein the beam is sufficiently collimated and a variationof an optical thickness of the sensor layer is such that a difference ofoptical paths inside the sensor layer for different light rays of saidbeam does not exceed a fourth of the smallest wavelength in the spectrumof said polychromatic light, wherein the polychromatic light has acoherence length that is less than double the distance between theboundary surfaces of the sensor layer; (e) recording a modulatedspectrum, containing interference maximums and minimums due tointerference either between light beams reflected from said boundarysurfaces with said substance and said material component or betweenlight beams transmitted through said boundary surfaces with saidsubstance and said material component; (f) tracking a spectral shift ofthe interference maximums and minimums of said modulated spectrum thatis due to a change of optical thickness between said boundary surfaceswith said substance because of binding with said material component; and(g) deriving from said spectral shift the parameters of binding of saidmaterial component to said substance.
 2. A method according to claim 1,wherein the sensor layer comprises a plurality of spatially separatedpieces each of which has a different surface orientation, each of saidspatially separated pieces being irradiated by light via fiber optics.3. A method according to claim 2, wherein said pieces are irradiated bysaid beam of polychromatic light simultaneously, and the modulatedspectrum is recorded for each of said pieces by spectrometers that arefurnished by optical fibers for input of light.
 4. A method according toclaim 3, wherein step (b) comprises immobilizing a plurality ofdifferent substances capable of selectively binding a plurality ofmaterial components on boundary surfaces of the spatially separatedpieces.
 5. A method according to claim 4, wherein the gaseous or liquidsample comprises the plurality of material components, the methodcomprising contacting the gaseous or liquid sample with at least one ofthe boundary surfaces of the spatially separated pieces, wherein step(g) comprises deriving parameters of bonding of each of said pluralityof material components to the plurality of different substances.
 6. Amethod according to claim 1, wherein the spectrum of said polychromaticlight possesses a width intrinsic to superluminescent laser diodes orlight-emitting diodes.
 7. A method according to claim 1, wherein thesensor layer comprises a plate with said two boundary surfaces notadjacent to any substrate.
 8. A method according to claim 7, whereinsaid substance is immobilized on a first boundary surface of the plate;said method comprising placing the liquid sample, which contains saidmaterial component, on the first boundary surface so that interactionbetween the material component and the substance takes place andcovering a second boundary surface of the plate with a coating thatprovides sufficient closeness of reflection coefficients of both thefirst and second boundary surfaces of the plate to reach a maximalcontrast of the interference maximums and minimums of the modulatedspectrum.
 9. A method according the claim 7, wherein said substance isimmobilized on a first boundary surface of the plate, said methodcomprising placing the liquid sample, which contains said materialcomponent, on the first boundary surface so that interaction between thematerial component and the substance takes place, and placing a liquidhaving a refractive index close to the refractive index of said liquidsample on a second boundary surface of the plate.
 10. A method accordingto claim 7, wherein said substance is immobilized on a first boundarysurface of the plate, said method comprising placing the liquid sample,which contains said material component, on both the first boundarysurface and on a second boundary surface of the plate.
 11. A methodaccording to claim 7, wherein step (b) comprises immobilizing aplurality of different substances capable of selectively binding aplurality of material components on a plurality of spatially separatedareas on said plate; and step (e) comprises recording modulatedspectrums for all of said plurality of spatially separated areas and forall wavelengths of said polychromatic light simultaneously byspectrometers.
 12. A method according to claim 1, wherein the sensorlayer comprises the two boundary surfaces with a gap therebetween, thetwo boundary surfaces comprising the solid optical material.
 13. Amethod according to claim 12, wherein step (b) comprises immobilizing aplurality of different substances capable of selectively binding aplurality of material components on a plurality of spatially separatedareas of said boundary surface; step (c) comprises filling said gap withthe gaseous sample or liquid sample containing said plurality ofmaterial components; step (e) comprises recording simultaneously allwavelengths of said modulated spectrum for each of said plurality ofspatially separated areas; step (f) comprises tracking a spectral shiftof said modulated spectrum for each of said plurality of spatiallyseparated areas; and step (g) comprises deriving the parameters ofbinding of each of said plurality of material components to saidplurality of different substances from said spectral shift for each ofsaid plurality of spatially separated areas.
 14. A method according toclaim 13, wherein the recording in step (e) is performed byspectrometers that are furnished by optical fibers for input of light.15. A method according to claim 13, wherein all of said areas areirradiated by all plurality of wavelengths of said polychromatic lightsimultaneously.
 16. A method according to claim 12, comprising placingan insertion in said gap between said boundary surfaces to split saidgap into a plurality of reaction cells including at least one reactioncell for use as a reference; said step (e) comprising recordingmodulated spectra for said plurality of reaction cells and for allplurality of wavelengths of said polychromatic light simultaneously byspectrometers.